KR101263294B1 - Antibodies against amyloid-beta peptide - Google Patents

Abstract

The present invention provides an antibody that binds to a human β-amyloid peptide, a method for treating a disease or condition characterized by increased β-amyloid levels or β-amyloid deposition with the antibody, a pharmaceutical composition comprising the antibody and a preparation thereof It is about a method.

Description

ANTIBODIES AGAINST AMYLOID-BETA PEPTIDE

The present invention relates to β-amyloid peptides and in particular to antibodies bound to human β-amyloid peptides. The present invention also relates to a method for treating a disease or condition, in particular Alzheimer's disease, characterized by increased β-amyloid levels or β-amyloid deposition with said antibody, a pharmaceutical composition comprising said antibody and a method for producing the same. Other aspects of the invention will be apparent from the following description.

Alzheimer's disease (AD) is the most common cause of age-related cognitive decline in more than 12 million individuals worldwide (Citron M (2002) Nat. Neurosci 5, Suppl 1055-1057). The earliest stages of the disease are gradual loss of memory and language and behavioral discomfort associated with decreased cognition. In later stages of the disease, the patient has a total memory loss and a very reduced motor function. Death usually occurs nine years after diagnosis and is often associated with other diseases, usually pneumonia (Davis KL and Samules SC (1998) in Pharmacological Management of Neurological and Psychiatric Disorders eds Enna SJ and Coyle JT (McGraw-Hill, New York) pp267-316). Current treatments are symptomatic approaches and focus on alleviating cognitive impairment and improving behavioral signs associated with ongoing disease etiology. Indeed, such treatments provide only short-lived cognitive benefits and cognitive impairment levels are reported to last only up to two years. There are numerous possibilities for disease-modifying therapies that slow or possibly stop the progression of the disease. This approach will not only provide fundamental and lasting improvements in the quality of life of patients and, importantly, caregivers, but will also reduce the overall enormous cost of managing this disease.

The clinical diagnosis of Alzheimer's disease is based on a combination of physical and mental tests that lead to possible or likely diagnosis of Alzheimer's disease. This disease, after death, is confirmed by well-characterized neurological validation in the brain, including deposition of Aβ in parenchymal plaques and cerebrovascular vessels, intracellular formation of neurofibrillary tangles, synaptic loss and nerves in certain brain regions. Loss of cell subpopulation (Terry, RD (1991) J Neural Trans Suppl 53: 141-145).

Numerous genetic, histological and functional evidence suggests that β-amyloid peptide (Aβ) is important in the progression of Alzheimer's disease (Selkoe, D. J. (2001) Physiological Reviews 81: 741-766).

Aβ is known to be produced through cleavage of a beta amyloid precursor protein (also known as APP) by an aspartyl protease enzyme known as BACE1 (also known as β-secretase, Asp2 or memacin-2) ( De Strooper, B. and Konig, G. (1999) Nature 402: 471-472). Soluble oligomeric forms of Αβ are assumed to contribute to the initiation of AD in addition to parenchymal and vascular deposition, which can initially affect neuronal function by impairing synaptic function (Lambert et. Al. (1998) Proceedings of the National Academy of Science, USA 95: 6448-6453. Although insoluble amyloid plaques are found early in AD and MCI, levels of soluble Αβ aggregates (referred to as oligomers or Αβ-derived diffuse ligands (ADDL)) also increase in these individuals, and soluble Αβ levels are more neuronal than amyloid plaques. More severely associated with fiber degeneration, and loss of synaptic markers (Naslund et. Al. (2000) J Am Med Assoc 283: 1571-1577, Younkin, S. (2001) Nat. Med. 1: 8-19) . The highly amyloidogenic Aβ42 and the amino-terminated truncated form Aβx-42 are prominent species of Aβ found in both diffuse and aged plaques (Iwatsubo, T (1994) Neuron . 13: 45-53, Gravina, SA (1995) J. Biol. Chem . 270: 7013-7016). Relative levels of Aβ42 appear to be important regulators of Aβ aggregation into amyloid plaques, and indeed Aβ42 has been shown to aggregate more readily than other Aβ forms in vitro (Jarrett, JT (1993) Biochemistry . 32: 4693- 4697) Aβ 42 has been considered as an initiation molecule in the pathogenesis of AD by itself (Younkin SG, (1998) J. Physiol . (Paris). 92: 289-292). Although Aβ42 is a small product of APP metabolism, but small changes in its production are associated with a large effect on Aβ deposition, it was considered that a reduction in Aβ42 alone could be an effective way to treat AD (Younkin SG, (1998) J. Physiol . (Paris). 92: 289-292). As evidence of this, mutations in amyloid precursor protein (APP) and presenilin genes have been reported to predominantly increase the relative levels of Aβ 42 and thereby shorten the onset of Alzheimer's disease (AD) (Selkoe DJ, Podlisny MB). (2002) Annu. Rev. Genomics Hum. Gemet. 3: 67-99). However, it should be noted that the deposition rate is also dependent on catabolism and Aβ clearance.

Animal models of amyloid deposition were generated by overexpressing mutant human transgenes in mice. Mice overexpressing a single human APP transgene typically develop cerebral plaque-like β-amyloid deposition from 12 months of age (Games D. et al., (1995) Nature 373: 523-527; Hsiao K. et al. , (1996) Science 274: 99-102)), mice carrying both mutant human APP and presenilin-1 (PS-1) transgenes typically have cerebral plaque-like β-amyloid at a two month early age. Deposition was developed (Kurt MA et al., (2001) Exp. Neurol. 171: 59-71; McGowan E. et al., (1999) Neurolbiol. Dis. 6: 231-244).

It is increasingly clear that the transport of exogenous Aβ between the central nervous system (CNS) and plasma plays a role in regulating brain amyloid levels (Shibata, et al (2000) J Clin Invest 106: 1489-1499), and CSF Aβ rapidly From CSF to plasma. Thus, active vaccination with Αβ peptide or passive administration of certain Αβ antibodies rapidly binds to peripheral Αβ and alters the dynamic equilibrium between plasma, CSF and ultimately the CNS. Indeed, at present there are a number of studies demonstrating that this approach can lower Aβ levels, reduce Aβ pathology and provide improved cognition in various transgenic models of amyloidosis. Limited studies in higher species have also been conducted. Caribbean Vervet monkeys (16-10 years old) were immunized with Αβ peptides over 10 months. Aβ40 levels increased 2-5 fold in peak plasma at 251 days, while CSF levels of Aβ40 and Aβ42 were significantly reduced by 100 days and then recovered towards baseline. This reduction in CSF is accompanied by a significant reduction in plaque burden (Lemere, CA (2004) Am J Pathology 165: 283-297). A similar increase in plasma Aβ levels was also detected after immunization of old (15-20 years old) Leus monkeys (Gandy, S (2004) Alzheimer Dis Assoc Disord 18:44:46).

The first immunotherapy targeting brain amyloid was AN-1792 from Elan / Wyeth, an active vaccine. This treatment ended after the development of clinical signs consistent with meningoencephalitis. Subgroup analyzes suggested that this treatment slowed the reduction of cognitive function (Nature Clin Pract Neurol (2005) 1: 84-85). Post hoc analysis of the patients also showed evidence of plaque-cleaning (Gilman S. et al, (2005) Neurology 64 (9) 1553-1562). Passive MAb therapy, Bapinizumab (AAB-001, Elan / Wyeth), has been shown to significantly improve cognitive scores in small phase I safety studies.

Other diseases or disorders characterized by increased β-amyloid levels or β-amyloid deposition include MCI, Blasko I (2006) Neurobiology of aging "Conversion from cognitive health to mild cognitive impairment and Alzheimer's disease: Prediction by plasma amyloid beta 42, medial temporal lobe atrophy and homocysteine "in press, e-published 19 Oct 2006), hereditary cerebral hemorrhage with Dutch type β-amyloidosis, cerebral β-amyloid angiopathy and various types of degenerative dementia, For example, those related to Parkinson's disease, progressive nuclear palsy, cortical basal degeneration, and diffuse Lewis type Alzheimer's disease (Mollenhauer B (2007) J Neural Transm e-published 23 Feb 2007, van Oijen, M Lancet Neurol. 2006 5: 655- 60) and Down syndrome (Mehta, PD (2007) J Neurol Sci. 254: 22-7).

Summary of the Invention

In one embodiment of the invention, an antibody or antigen thereof that binds to β-amyloid peptide 1-12 (SEQ ID NO: 15) but not to β-amyloid peptide 2-13 (SEQ ID NO: 44) with an equilibrium constant KD of less than 100 pM There are provided therapeutic antibodies that are binding fragments and / or derivatives, both measurements being performed in a surface plasmon resonance assay using peptides captured on streptavidin chip.

In another embodiment of the invention, an equilibrium constant KD of less than 100 pM is bound to β-amyloid peptide 1-12 (SEQ ID NO: 15) and an equilibrium constant KD for binding to peptide 1-12 (SEQ ID NO: 15) A therapeutic antibody is provided that has an equilibrium constant KD or an antigen binding fragment and / or derivative thereof that relates to binding to β-amyloid peptide 2-13 (SEQ ID NO: 44)> 1000 fold, both measurements being strep Surface plasmon resonance assays using peptides captured on the tabidine chip are performed.

In another embodiment of the invention, an equilibrium constant KD of less than 100 pM is bound to β-amyloid peptide 1-12 (SEQ ID NO: 15) and an equilibrium constant KD for binding to peptide 1-12 (SEQ ID NO: 15) A therapeutic antibody is provided that has an equilibrium constant KD or an antigen binding fragment and / or derivative thereof that relates to binding to β-amyloid peptide 2-13 (SEQ ID NO: 44)> 10,000 fold, both measurements being strep Surface plasmon resonance assays using peptides captured on the tabidine chip are performed.

In one aspect, the surface plasmon resonance assay using peptides captured on streptavidin chips is the surface plasmon resonance assay described in the Examples below. In another aspect, the surface plasmon resonance assay using a peptide captured on a streptavidin chip is Method A (i) disclosed in the following SPR Biacore ™ assay.

In an alternative embodiment of the invention, an antibody or antigen-binding fragment thereof which binds to β-amyloid peptide 1-40 but not to β-amyloid peptide 2-13 (SEQ ID NO: 44) with an equilibrium constant KD of less than 10 nM and / Or a therapeutic antibody which is a derivative, wherein both measurements are performed with a surface plasmon resonance assay described in Method B of the Examples below.

In another alternative embodiment of the invention, the β-amyloid peptide 1-40 is bound with an equilibrium constant KD of less than 10 nM and the equilibrium constant KD for binding to peptides 1-12 (SEQ ID NO: 15) is greater than 1000 times There is provided a therapeutic antibody which is an antibody or an antigen-binding fragment and / or derivative thereof having an equilibrium constant KD for binding to β-amyloid peptide 2-13 (SEQ ID NO: 44), wherein both measurements are methods of the Examples The surface plasmon resonance assay described in B is performed.

In another alternative embodiment of the invention, the β-amyloid peptide 1-40 is bound with an equilibrium constant KD of less than 10 nM and the equilibrium constant KD for binding to peptides 1-12 (SEQ ID NO: 15) is greater than 10,000 times There is provided a therapeutic antibody which is an antibody or an antigen-binding fragment and / or derivative thereof having an equilibrium constant KD for binding to β-amyloid peptide 2-13 (SEQ ID NO: 44), wherein both measurements are methods of the Examples The surface plasmon resonance assay described in B is performed.

In an embodiment of the invention, there is provided a therapeutic antibody which is an antibody or antigen-binding fragment and / or derivative thereof that binds to the β-amyloid peptide and comprises the following CDRs:

In a human heavy chain variable region derived from the VH3 gene family

CDRH1: DNGMA (SEQ ID NO: 1)

CDRH2: FISNLAYSIDYADTVTG (SEQ ID NO: 2)

CDRH3: GTWFAY (SEQ ID NO: 3) and:

Within a human light chain variable region derived from the amino acid sequence set forth in GenPept entry CAA51135 (SEQ ID NO: 24).

CDRL1: RVSQSLLHSNGYTYLH (SEQ ID NO: 4)

CDRL2: KVSNRFS (SEQ ID NO: 5)

CDRL3: SQTRHVPYT (SEQ ID NO: 6).

In this specification, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2" and "CDRH3" are described in Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987] according to the Kabat numbering system. Thus, the following defines a CDR according to the present invention:

CDR: residue

CDRH1: 31-35B

CDRH2: 50-65

CDRH3: 95-102

CDRL1: 24-34

CDRL2: 50-56

CDRL3: 89-97

VH3 gene family and related immunoglobulin gene nomenclature are disclosed in Matsuda et al (Journal of Experimental Medicine, 188: 2151-2162, 1998) and Lefranc & Lefranc (The Immunoglobulin Factsbook. 2001. Academic Press: London). .

In certain embodiments, the human heavy chain variable region is derived from:

V gene selected from among VH3 members of the following subsets: VH3-48, VH3-21, VH3-11, VH3-7, VH3-13, VH3-74, VH3-64, VH3-23, VH3-38, VH3-53, VH3-66, VH3-20, VH3-9 and VH3-43.

V gene selected from the VH3 family members of the following subsets: VH3-48, VH3-21 and VH3-11.

The VH3-48 gene, or allele thereof.

The sequence of the Genbank entry M99675 is an allele of the VH3-48 gene. M99675 is a Genbank nucleotide sequence of a genomic piece of DNA comprising two exons that constitute the human heavy chain gene VH3-48 (SEQ ID NO: 22) and encode the variable region amino acid sequence set forth in SEQ ID NO: 21. In certain aspects, the human receptor heavy chain framework is derived from M99675.

Framework 4 should be added to germline encoded V-gene M99675 to construct the complete V-region. Suitable framework 4 sequence YFDYWGQGTLVTVSS (SEQ ID NO: 23) includes those encoded by the human JH4 minigene (Kabat):

Its first four residues are in the CDR3 region replaced by the transgenic CDRs from the donor antibody.

Those skilled in the art understand that germ cell V gene and J gene do not comprise coding sequences for the entire heavy chain CDR3. However, in the antibodies of the invention, the CDR3 antibodies are provided by donor immunoglobulins. Thus, the combination of a VH gene such as VH3-48, a JH minigene such as JH4, and a set of heavy chain CDRs such as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 mimics a mature fully rearranged heavy chain variable region Assembled in such a way) that is sufficient to define a heavy chain variable region of the invention as represented by SEQ ID NOs: 26, 28, 30.

The variable region encoded by Genpept ID CAA51134 has the amino acid sequence provided as SEQ ID NO: 24.

The light chain variable region framework sequence known as GenPept ID CAA51134 is an inferred amino acid sequence of the fully rearranged light chain variable region and is identical to another amino acid sequence having the same framework in the database: Genpept accession no. S40356, Klein, R., et al., Eur. J. Immunol. 23 (12), 3248-3262 (1993). The DNA coding sequence for CAA51134, available as Genbank Accession No. X72467, is provided as SEQ ID NO: 25.

In certain aspects of the invention, the human receptor heavy chain framework is derived from the M99675 and JH4 minigenes and the human receptor light chain framework contains substitutions of one or more, such as from 1 to 4, more preferably from 1 to 3 amino acid residues. Derived from or without CAA51135, which comprises a donor V _H domain having a sequence of SEQ ID NO: 17 that retains all binding affinity or substantially all binding affinity of a donor antibody to a β-amyloid peptide Is based on the corresponding residue found in the V _L domain.

"Substantially all binding affinity" means that the binding affinity of a therapeutic antibody is at most 10-fold less than that of a donor antibody.

In more particular aspects, the human receptor heavy chain framework derived from M99675 and JH4 has 1 to 4 amino acid residue substitutions selected from positions 24, 48, 93 and / or 94 (Kabat numbering).

In a more particular aspect of the invention, the human receptor heavy chain framework derived from M99675 and JH4 comprises the following residues (or conservative substituents thereof).

(i)

location Residue

93 V

94 S

or

(ii)

location Residue

24 V

93 V

94 S

or

(iii)

location Residue

48 I

93 V

94 S

In one embodiment of the invention, there is provided a therapeutic antibody which is an antibody comprising a V _H chain having the sequence set forth in SEQ ID NO: 26, 28 or 30 and a V _L domain having the sequence set forth in SEQ ID NO: 32.

In another embodiment of the invention, there is provided a therapeutic antibody which is an antibody comprising a heavy chain having the sequence set forth in SEQ ID NO: 34, 36 or 38 and a light chain having the sequence set forth in SEQ ID NO: 40.

In another embodiment of the invention, there is provided a pharmaceutical composition comprising a therapeutic antibody according to the invention.

In a further embodiment of the present invention there is provided a method of treating a patient, comprising administering a therapeutically effective amount of a therapeutic antibody according to the invention to a human patient with a β-amyloid peptide-related disease.

Also provided is the use of a therapeutic antibody according to the invention in the manufacture of a medicament for treating a β-amyloid peptide-related disease.

In another embodiment of the invention, there is provided a method of preparing a therapeutic antibody according to the invention comprising expressing in a host cell a polynucleotide encoding the antibody.

In another embodiment of the present invention, a polynucleotide is provided that encodes a therapeutic antibody heavy chain comprising a V _H chain having the sequence set forth in SEQ ID NO: 26, 28 or 30.

In another embodiment of the invention, a polynucleotide is provided that encodes a therapeutic antibody light chain comprising a V _L domain having the sequence set forth in SEQ ID NO: 32.

In another embodiment of the present invention, a polynucleotide is provided that encodes a therapeutic antibody heavy chain having the sequence set forth in SEQ ID NO: 34, 36 or 38.

In another embodiment of the present invention, a polynucleotide is provided which encodes a therapeutic antibody light chain having the sequence set forth in SEQ ID NO: 40.

In a more particular embodiment of the present invention, a polynucleotide is provided which encodes a therapeutic antibody heavy chain comprising the sequence set forth in SEQ ID NO: 35, 37, 39 or 42.

In another more specific embodiment of the present invention, a polynucleotide is provided that encodes a therapeutic antibody light chain comprising the sequence set forth in SEQ ID NO: 41 or 43.

In certain embodiments, the therapeutic antibody, which is the antibody or fragment and / or derivative thereof, essentially lacks the ability to a) activate complement by the standard route and b) mediate the cytotoxicity of antibody-dependent cells.

In another embodiment of the present invention, an antibody or fragment thereof is provided comprising a V _H domain having a sequence of SEQ ID NO: 17 and a V _L domain having a sequence of SEQ ID NO: 19.

In another embodiment of the invention, there is provided a polynucleotide, in particular a polynucleotide encoding SEQ ID NO: 18, comprising an antibody heavy chain or fragment thereof comprising a V _H domain having a sequence of SEQ ID NO: 17.

In another embodiment of the invention, there is provided a polynucleotide encoding an antibody light chain or fragment thereof comprising a V _L domain having the sequence of SEQ ID NO: 19, in particular a polynucleotide of SEQ ID NO: 20.

DETAILED DESCRIPTION OF THE INVENTION

1. Antibody Structure

1.1 Complete Antibody

Complete antibodies are generally heteromultimeric glycoproteins comprising two or more heavy chains and two light chains. Except for IgM, a complete antibody is a heterodimeric glycoprotein of about 150 KDa consisting of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is bound to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has a disulfide bridge in the chain. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant regions. Each light chain has at its other end a variable domain (V _L ) and a constant region; The constant region of the light chain is aligned with the first constant region of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Light chains of antibodies from most vertebrate species can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of the constant region. Depending on the amino acid sequences of the constant regions of their heavy chains, human antibodies can be assigned to five different classes of IgA, IgD, IgE, IgG and IgM. IgG and IgA are subclasses IgG1, IgG2, IgG3 and IgG4; And IgA1 and IgA2. Species variants are present in mice and rats having at least IgG2a, IgG2b. The variable domain of an antibody confers binding specificity for the antibody with specific regions exhibiting specific variability called complementarity determining regions (CDRs). The more conserved portions of the variable regions are called framework regions FR. The variable domains of the complete heavy and light chains each comprise four FRs linked by three CDRs. The CDRs of each chain are kept in close proximity to each other by the FR regions and contribute to the formation of the antigen binding site of the antibody with the CDRs from the other chain. The constant region is not directly involved in binding the antibody to the antigen but is antibody dependent cell-mediated cytotoxicity (ADCC), phagocytosis through binding to Fcγ receptors, half-life / cleaning rate through neonatal Fc receptor (FcRn) and complement It exhibits a variety of effector functions such as those involved in complement dependent cytotoxicity through the C1q component of the cascade. Human IgG2 constant regions lack the ability to activate complement or mediate cytotoxicity of antibody-dependent cells by traditional pathways. IgG4 constant regions lack the ability to activate complement by traditional pathways and only mediate weakly the cytotoxicity of antibody-dependent cells. Antibodies that essentially lack this effector function may be referred to as "non-cytolytic" antibodies.

1.1.1 Human Antibodies

Human antibodies can be produced by a number of methods known to those skilled in the art. Human antibodies can be prepared by hybridoma methods using human myeloma or mouse-human heteromyeloma cell lines. See Kozbor J. Immunol 133, 3001, (1984) and Brodeur, Monoclonal Antibody Production Techniques and Applications, pp 51-. 63 (Marcel Dekker Inc, 1987) An alternative method is the use of phage libraries or transgenic mice using the human V region repertoire [Winter G, (1994), Annu. Rev. Immunol 12,433-455, Green. LL (1999), J. Immunol.methods 231, 11-23.

Several strains of transgenic mice are currently available, where their mouse immunoglobulin loci have been replaced with human immunoglobulin gene segments (see Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature Biotechnol. 14,845-851, Mendez MJ, 1997, Nature Genetics, 15,146-156. Upon antigen challenge, such mice can generate a repertoire of human antibodies from which the antibody of interest can be selected.

Of particular note is the Trimera ™ system (Eren R et al, (1998) Immunology 93: 154-161), which transplants human lymphocytes into irradiated mice, in which a large amount of human (or other species) lymphocytes were pooled. Selected lymphocyte antibody systems (SLAM, see Babcook et al, PNAS (1996) 93: 7843-7848) and Xenomouse that are subjected to in vitro antibody development procedures and deconvulated while limiting dilution and selection processes. II ™ (Abgenix Inc). Alternative approaches can be used from Morphotek Inc using Morphodoma ™ technology.

Phage display technology can be used to generate human antibodies (and fragments thereof) (see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD et al . (1994) EMBO 13: 3245-3260. According to this technique, the antibody V domain gene is cloned into the major or minor envelope of a protein gene of a fibrous bacteriophage such as M13 or fd in a frame and displayed as a functional antibody fragment on the surface of the phage particle (generally with helper phage help). Received). Selection based on the functionality of the antibody selects the gene encoding the antibody exhibiting such properties. Phage display techniques can be used to select antigen specific antibodies from libraries prepared from human B cells obtained from an individual suffering from the disease or condition disclosed above or alternatively from an unimmunized human donor (see Marks; J. Mol. Bio. 222,581-597, 1991]. If it is desired for a fully human antibody to comprise an Fc domain, it is essential to reclones the phage displayed derived fragment into a mammalian expression vector that contains the desired constant region and establishes a stable expression cell line.

Affinity maturation techniques (Marks; Bio / technol 10,779-783 (1992)) can be used to improve binding affinity, where the affinity of primary human antibodies in turn replaces the H and L chain V regions with naturally occurring variants It is improved by selecting based on improved binding affinity. Variants of this technique such as "epitope imprinting" are currently available (see WO 93/06213). See also Waterhouse; Nucl. Acids Res 21, 2265-2266 (1993).

1.2 Chimeric and Humanized Antibodies

The use of a complete non-human antibody in the treatment of a human disease or disease entails the possibility for a well-established problem of immunogenicity, in particular in the repeated administration of the antibody, i.e. the patient's immune system is a non-human antibody. It can be recognized as a non-self antibody and ready for neutralization reaction. In addition to developing fully human antibodies (see above), various techniques have been developed over the years to overcome these problems and are generally used in immunized animals, for example, while reducing the composition of non-human amino acid sequences in fully therapeutic antibodies, Retaining relative ease in obtaining non-human antibodies from mice, rats or rabbits. Broadly, two approaches have been used to achieve this. The first is a chimeric antibody that generally comprises a non-human (eg rodent such as mouse) variable domain fused to a human constant region. Since the antigen-binding site of the antibody is localized in the variable region, the chimeric antibody retains its binding affinity for the antigen, and can acquire effector functions of the human constant region to perform effector functions as described above. Chimeric antibodies are typically generated using recombinant DNA methods. DNA encoding the antibody (e.g., cDNA) is a conventional process (e.g., a gene encoding the H and L chain variable regions of an antibody of the invention, e.g., DNAs of SEQ ID NOs: 18 and 20 described above). By using an oligonucleotide probe that can specifically bind to). Hybridoma cells serve as a typical source of such DNA. Once separated, DNA is located in the expression vector, followed by the immunoglobulin host cell, for example, Escherichia coli that does not produce the protein (E.Coli) for achieving the synthesis of the antibody, COS cells, CHO cells, PerC6 cells or myeloma cells Transfected. DNA can be modified by replacing the coding sequences for human L and H chains with the corresponding non-human (eg murine) H and L constant regions (see, eg, Morrison; PNAS 81, 6851 ( 1984). Thus, in another embodiment of the invention comprising a V _L domain having the sequence of SEQ ID NO: 19 fused to a human constant region (which may be an IgG isotype such as IgG1) and a V _H domain having the sequence of SEQ ID NO: 17 Chimeric antibodies are provided.

The second approach involves the production of humanized antibodies, wherein the non-human content of the antibodies is reduced by humanizing the variable regions. Two techniques for humanization are popular. The first is humanization by CDR grafting. The CDRs establish a loop close to the N-terminus of the antibody, where they form a surface that fits into the backbone provided by the framework regions. Antigen-binding specificity of an antibody is mainly defined by the chemical properties and topography of its CDR surface. These features are then determined by the shape of the individual CDRs, the relative placement of the CDRs, and the nature and placement of the side chains of residues comprising the CDRs. Large reductions in immunogenicity can only be achieved by grafting the CDRs of non-human (eg, murine) antibodies (“donor” antibodies) into suitable human frameworks (“receptor frameworks”) and constant regions. (See Jones et al (1986) Nature 321, 522-525 and Verhoeyen M et al (1988) Science 239, 1534-1536). However, CDR grafting itself may not fully maintain antigen-binding properties, and when significant antigen binding affinity is restored, some framework residues of the donor antibody may be preserved in the humanized molecule (sometimes referred to as "return mutations" backmutations. ”(Queen C et al . (1989) PNAS 86, 10,029-10,033, Co, M et al . (1991) Nature 351, 501-502). In such cases, the human V region that exhibits the greatest sequence homology (typically 60% or more) for non-human donor antibodies can be selected from the database to provide a human framework (FR). The selection of human FRs can be made from human consensus or individual human antibodies. If necessary, the necessary major residues from the donor antibody are replaced with a human acceptor framework to preserve the CDR morphology. Computer modeling of the antibodies can be used to help identify these structurally important residues (see WO99 / 48523).

Alternatively, humanization can be accomplished by a "veneering" process. By statistical analysis of unique human and murine immunoglobulin heavy and light chain variable regions, the exact pattern of exposed residues differs between human and murine antibodies, with most individual surface positions having a strong preference for fewer different residues (Padlan EA et al . (1991) Mol. Immunol. 28, 489-498 and Pedersen JT et al . (1994) J. Mol. Biol. 235; 959-973). Reference). Thus, it is possible to lower the immunogenicity of non-human Fv by replacing exposed residues in framework regions that are different from those commonly found in human antibodies. Since protein antigenicity may be related to surface accessibility, substitution of surface residues may be sufficient to allow the "invisible" mouse variable region to bear the human immune system (Mark GE et al (1994) in Handbook of Experimental Pharmacology vol. 113: The pharmacology of monoclonal Antibodies , Springer-Verlag, pp 105-134). This process of humanization is called "veneering" because only the surface of the antibody is altered and the support residues remain undisturbed. Further alternative approaches include the methods disclosed in WO04 / 006955 and the Huminering ™ procedure (Kalobios), which produces antibodies intimate with human germ cells in sequences using bacterial expression systems (Alfenito-M Advancing). Protein Therapeutics January 2007, San Diego, California).

It will be apparent to those skilled in the art that the term "derived" is intended to define not only a source in terms of physical origin for the substance, but also a substance that is structurally identical to the substance but not derived from a reference source. Thus, the "residues found in the donor antibody" do not necessarily have to be purified from the donor antibody.

It will be apparent to one skilled in the art that certain amino acid substitutions are considered to be "conservative." Amino acids are divided into groups based on common side chain properties, and substitutions within the group that retain all or substantially all of the binding affinity of the therapeutic antibody of the invention are considered conservative substitutions. See Table 1 below:

Table 1

chain member Hydrophobic met, ala, val, leu, ile Neutral hydrophilic cys, ser, thr acid asp, glu Basic asn, gln, his, lys, arg Residues Affecting Chain Orientation gly, pro Aromatic trp, tyr, phe

1.3 bispecific antibodies

Bispecific antibodies are antibody derivatives having binding specificities for at least two different epitopes and form part of the present invention. Methods of making such antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin H chain-L chain pairs, where the two H chains have different binding specificities (Millstein et al, Nature 305 537-). 539 (1983), WO 93/08829 and Traunecker et al EMBO, 10, 1991, 3655-3659). Random sorting of the H and L chains results in a potential mix of ten different antibody structures, only one of which has the desired binding specificity. An alternative method involves fusing a variable domain having a desired binding specificity to a heavy chain constant region comprising at least some of the hinge region, CH2 and CH3 regions. It is preferred to have a CH1 region containing the site required for light chain binding present in one or more of the fusions. DNA encoding this fusion, and if necessary, the L chain is inserted into a separate expression vector and then cotransfected into a suitable host organism. It is possible to insert the coding sequences for two or all three chains into one expression vector. In one preferred approach, the bispecific antibody consists of an H chain with the first binding specificity in one arm and HL chain pair, and the second binding specificity is present in the other cancer. See WO94 / 04690. See also Suresh et al . Methods in Enzymology 121, 210, 1986.

Delivery of therapeutic proteins to the brain has been hampered by the presence of the blood brain barrier (BBB). Where it is desired to deliver an antibody of the invention or an antibody fragment of the invention across a BBB, various methods have been proposed to enhance such delivery if necessary.

To obtain the nutrients and factors required from the blood, BBB possesses some specific receptors, which carry compounds from the circulating blood to the brain. Some compounds, such as insulin, from studies have been described by Duffy KR et al . (1989) Brain Res. 420: 32-38], transferrin (see Fishman JB et al . (1987) J. Neurosci 18: 299-304] and insulin-like growth factors 1 and 2 [see, Pardridge WM (1986) Endocrine Rev. 7: 314-330 and Duffy KR et al. (1986) Metabolism 37: 136-140 has been pointed out across BBB through receptor-mediated transcytosis. Thus, receptors for these molecules provide a potential means for the therapeutic antibodies of the invention to access the brain using so-called "vectored" antibodies (see Pardridge WM (1999) Advanced Drug). Delivery Review 36: 299-321). For example, antibodies to the transferrin receptor have been shown to be dynamically delivered to the brain parenchyma (Friden PM et al (1991) PNAS 88: 4771-4775 and Friden PM et al (1993) Science 259: 373-377). . Thus, one potential approach is bispecific as described above where the first specificity is for the transport receptor and the second specificity is for the transport receptor located in the BBB, eg, the second specificity is for the transferrin transport receptor. To provide an antibody or bispecific fragment.

1.4 Antibody Fragments

In certain embodiments of the invention, therapeutic antibodies are provided that are antigen binding fragments. Such fragments may be functional antigen binding fragments of complete and / or humanized and / or chimeric antibodies such as Fab, Fd, Fab ', F (ab') ₂ , Fv, ScFv fragments of the antibodies described above. Fragments lacking constant regions lack the ability to activate complement or mediate cytotoxicity of antibody-dependent cells by traditional pathways. Typically, such fragments are produced, for example, by proteolysis of the complete antibody by papain digestion (see eg WO94 / 29348) and can be produced directly from recombinantly transformed host cells. For the generation of ScFv, see Bird et al; (1988) Science, 242, 423-426. In addition, antibody fragments can be generated using various engineering techniques, as described below.

Fv fragments are shown to have lower interaction energy of their two chains than Fab fragments. To stabilize the binding of the V _H and V _L domains, these are peptides (Bird et al, (1988) Science 242, 423-426, Huston et al., PNAS, 85, 5879-5883), disulfide bonds (Glockshuber et al. , (1990) Biochemistry, 29, 1362-1367) and "knob in hole" mutations (Zhu et al (1997), Protein Sci., 6, 781-788). ScFv fragments can be generated by methods well known to those skilled in the art (Whitlow et al (1991) Methods companion Methods Enzymol, 2, 97-105 and Huston et al (1993) Int. Rev. Immunol 10, 195- 217). ScFv may be produced in bacterial cells such as E. Coli , and more preferably in eukaryotic cells. One disadvantage of ScFv is in monovalent products, which excludes the increased antigen-binding ability and their short half-life due to multivalent binding. Attempts to overcome these problems include chemical coupling (Adams et al (1993) Can. Res 53, 4026-4034 and McCartney et al (1995) Protein Eng. 8, 301-314) or unpaired C-terminal cysteines. Divalent (ScFv ′) ₂ generated from ScFV containing additional C-terminal cysteines by spontaneous site specific dimerization of ScFv containing residues (Kipriyanov et al (1995) Cell. Biophys 26, 187- 204). Alternatively, ScFv can shorten the peptide linker between 3 and 12 residues to form multimers to form "diabodies" (Holliger et al PNAS (1993), 90, 6444-). 6448). Reducing the linker is further described by the ScFV trimer ("Tribody", see Kortt et al (1997) Protein Eng, 10, 423-433) and tetramers ("Tetrabody", Le Gall et al. (1999) FEBS Lett, 453, 164-168). The construction of divalent ScFV molecules is also accomplished by genetic fusion with protein dimerization motifs to allow for "miniantibodies" (see Pack et al (1992) Biochemistry 31, 1579-1584) and "minibodies" (see literature). (See Hu et al (1996), Cancer Res. 56, 3055-3061). ScFv-Sc-Fv tandem ((ScFV) ₂ ) can also be generated by linking two ScFv units by a third peptide linker (Kurucz et al (1995) J. Imol. 154, 4576-4582). ] Reference). Bispecific diabodies can be produced via monovalent binding of two single chain fusion products consisting of a V _H domain from another antibody linked by a short linker to the V _L domain of one antibody (Kipriyanov et al ( 1998), Int. J. Can 77, 763-772). The stability of such bispecific diabodies is enhanced by influx of disulfide bridges or “knob in hole” mutations as described above or by the formation of single chain diabodies (ScDb) in which two hybrid ScFv fragments are linked via a peptide linker. (See Kontermann et al (1999) J. Immunol. Methods 226 179-188). Trivalent bispecific molecules are available, for example, by fusing a ScFv fragment through a hinge region to a CH3 domain or Fab fragment of an IgG molecule (see Coloma et al (1997) Nature Biotechnol. 15, 159-163). ). Alternatively, the tetravalent bispecific molecules were produced by fusion of bispecific single chain diabodies (see Alt et al, (1999) FEBS Lett 454, 90-94). Smaller saga bispecific molecules may also be used in the ScFv-ScFv tandem (see DiBi miniantibody, Muller et al (1998) FEBS Lett 432, 45-49) with a linker containing a helix-loop-helix motif, or Single chain molecules containing four antibody variable domains (V _H and V _L ) in an orientation that prevents intramolecular pairing (tandem diabodies, Kipriyanov et al, (1999) J. Mol. Biol. 293, 41- 56). Bispecific F (ab ') ₂ fragments can be generated through chemical coupling of Fab' fragments or through heterodimerization via leucine zippers (Shalaby et al, (1992) J. Exp. Med. 175, 217-225 and Kostelny et al (1992), J. Immunol. 148, 1547-1553). Separate V _H and V _L domains are also available (see US 6,248,516; US 6,291,158; US 6,172,197).

1.5 Heteroconjugate Antibodies

Heteroconjugate antibodies are also derivatives which form embodiments of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies formed using any convenient crosslinking method. See, for example, US 4,676,980.

1.6 Other Modifications

Interactions between the Fc region of antibodies and various Fc receptors (FcγRs) mediate effector functions of antibodies, including antibody-dependent cellular cytotoxicity (ADCC), complement fixation, phagocytosis, and antibody half-life / cleaning rate. Is considered. Various modifications to the Fc region of the antibodies of the invention can be performed depending on the desired effector properties. Specifically, human constant regions that a) activate complement by traditional pathways and b) essentially lack the ability to mediate the cytotoxicity of antibody-dependent cells include IgG4 constant regions, IgG2 constant regions and IgG1 constant regions; These include the specific mutations as mutations at, for example, positions 234, 235, 236, 237, 297, 318, 320 and / or 322 described in EP0307434 (WO8807089), EP 0629 240 (WO9317105) and WO 2004/014953. It contains. Mutations at residues 235 or 237 in the CH2 domain of the heavy chain constant region (Kabat numbering; EU Index system) reduce binding to FcγRI, FcγRII and FcγRIII, respectively, reducing antibody-dependent cellular cytotoxicity (ADCC) (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51; 1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168. In addition, some reports describe the involvement of some of these residues in recruiting or mediating complement dependent cytotoxicity (CDC) (Morgan et al., 1995; Xu et al., Cell. Immunol. 2000; 200; 200 : 16-26; Hezareh et al., J. Virol. 2001, 75 (24); 12161-12168). Accordingly, both residues 235 and 237 were mutated to alanine residues (Brett et al. Immunology 1997, 91; 346-353; Bartholomew et al. Immunology 1995, 85; 41-48; and WO9958679) with complement mediated effects. Both FcγR-mediated effects were reduced. Antibodies comprising such constant regions may be referred to as "non-cytolytic" antibodies.

One skilled in the art can incorporate salvage receptor binding epitopes into antibodies to increase serum half-life (see US 5,739,277).

There are five currently recognized human Fcγ receptors, FcγR (I), FcγRIIa, FcγRIIb, FcγRIIIa and neonatal FcRn. Shields et al, (2001) J. Biol. Chem 276, 6591-6604 show that a common set of IgG1 residues is involved in the binding of all FcγRs, while FcγRII and FcγRIII use distinct sites other than this common set. Proved. When one of the IgGl residues is changed to alanine, it lowers binding to all FcγRs: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239. All are located in the IgG CH2 domain and clump near the hinge that binds CH1 and CH2. Only FcγRI uses a common set of IgG1 residues for binding, while FcγRII and FcγRIII interact with distinct residues other than the common set. Alteration of some residues only degraded binding to FcγRII (eg Arg-292) or FcγRIII (eg Glu-293). Some variants show increased binding to FcγRII or FcγRIII, but do not affect binding to other receptors (eg, Ser-267Ala enhances binding to FcγRII but not to FcγRIII). ). Other variants exhibit enhanced binding to FcγRII or FcγRIII, and binding to other receptors is reduced (eg, Ser-298Ala enhances binding to FcγRIII but decreases binding to FcγRII). For FcγRIIIa, the best binding IgG1 variants were combined with alanine substituents at Ser-298, Glu-333 and Lys-334. Neonatal FcRn receptors are believed to be involved in protecting IgG molecules from degradation, enhancing serum half-life and transcytosis across tissues (Junghans RP (1997) Immunol. Res 16. 29-57 and Ghetie et al ( 2000) Annu. Rev. Immunol. 18, 739-766. Human IgGl residues determined to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435.

The therapeutic antibody of the invention may comprise any of the above constant region modifications.

In certain embodiments, the therapeutic antibody essentially lacks the ability to a) activate complement by traditional pathways and b) mediate the cytotoxicity of antibody-dependent cells. In more particular embodiments, the present invention provides a therapeutic antibody of the invention having any one (or more) of the residue changes described above to provide half-life / cleaning rate and / or effector function such as ADCC and / or complement dependent cytotoxicity. And / or complement dissolution.

In a further aspect of the invention, the therapeutic antibody is isotype with an alanine (or other disruption) substituent at positions 235 (eg L235A) and 237 (eg G237A) (numbering according to the EU scheme outlined in Kabat). Has a constant region of human IgG1.

Other derivatives of the invention include glycosylation variants of the antibodies of the invention. Glycosylation of antibodies at positions conserved in their constant regions is known to significantly affect antibody action, particularly effector action as described above (see, eg, Boyd et al (1996), Mol. Immunol. 32, 1311-1318). Glycosylated variants of the therapeutic antibody of the invention wherein one or more carbohydrate moieties are added, substituted, deleted or modified are contemplated. Influx of asparagine-X-serine or asparagine-X-threonine motifs creates a potential site for enzyme attachment of carbohydrate moieties and thus can be used to manipulate glycosylation of antibodies. In Raju et al (2001) Biochemistry 40, 8868-8876, terminal sialylation of TNFR-IgG immunoadhesin is characterized by beta-1,4-galactosyltransferase and / or alpha, 2,3 sialyltransfer. Increased through the process of regalactosylation and / or resialylation using laase. Increased terminal sialylation is believed to increase the half-life of the immunoglobulin. Antibodies, along with most glycoproteins, are typically produced in nature as a mixture of glycoforms. Such a mixture becomes particularly evident when the antibody is produced in eukaryotes, in particular mammalian cells. Various methods have been developed for preparing defined sugar forms (Zhang et al Science (2004), 303, 371, Sears et al, Science, (2001) 291, 2344, Wacker et al (2002) Science, 298 1790). , Davis et al (2002) Chem. Rev. 102, 579, Hang et al (2001) Acc. Chem. Res 34, 727). As such, the present invention is directed to a method as described herein, comprising a defined number (eg, 7 or less, such as 5 or less, such as 2 or 1) of the glycoform (s) of the antibody. It relates to a number of therapeutic antibodies (possibly of IgG isotype eg IgG1).

Derivatives according to the invention also comprise therapeutic antibodies of the invention coupled to nonproteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylenes. Protein conjugation to PEG is an established technique that increases the half-life of a protein and reduces the antigenicity and immunogenicity of the protein. The use of PEGylation with different molecular weights and styles (linear or branched) has been investigated with complete antibodies and Fab 'fragments (see Komenis IL et al (2000) Int. J. Pharmaceut. 198: 83-95). . Certain embodiments include antigen-binding fragments of the invention (eg, Fab fragments or scFv) coupled to PEG, which a) activate complement by traditional pathways and b) lack effector functions that mediate the cytotoxicity of antibody-dependent cells. ).

2. How to create

Antibodies of the invention include transgenic organisms such as goats (see Pollock et al (1999), J. Immunol. Methods 231: 147-157), chickens (Morrow KJJ (2000) Genet. Eng. News 20 : 1-55), mice (see Pollock et al) or plants (Doran PM, (2000) Curr. Opinion Biotechnol. 11, 199-204, Ma JK-C (1998), Nat. Med. 4; 601-606, Baez J et al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000) Plant Mol. Biol. 42: 583-590). Antibodies can also be generated by chemical synthesis. However, the antibodies of the invention are typically produced using recombinant cell culture techniques known to those skilled in the art. The polynucleotide encoding the antibody is isolated and inserted into a replicable vector such as a plasmid for proliferation or expression in a host cell. One useful expression system is the glutamate synthetase system (such as sold by Lonza Biologies), in particular where the host cell is CHO or NS0 (see below). Polynucleotides encoding an antibody are readily isolated and sequenced using conventional processes (eg, oligonucleotide probes). Vectors that can be used include plasmids, viruses, phages, transposons, minichromosomes, of which plasmids are typical embodiments. In general, such vectors further include transcription termination sequences, promoters, enhancer elements, one or more marker genes, origins of replication, and signal sequences that are operably linked to light and / or heavy chain polynucleotides to promote expression. Polynucleotides encoding light and heavy chains can be inserted into separate vectors and introduced (eg, transformed, transfected, electroporated or transduced) simultaneously or sequentially into the same host cell, or, if desired, Both light chains can be inserted into the same vector prior to such introduction.

Due to the duplication of the genetic code, it will soon be apparent that polynucleotides which will encode polypeptides of the invention, which are alternatives to the polynucleotides described herein, are also available.

2.1 signal sequence

Antibodies of the invention can be produced as fusion proteins with heterologous signal sequences having specific cleavage sites at the N terminus of the mature protein. The signal sequence must be able to be recognized and processed by the host cell. For prokaryotic host cells, the signal sequence may be an alkaline phosphatase, penicillinase, or a heat stable enterotoxin II leader. In yeast secretion, the signal sequence can be a yeast convertase leader, a factor leader or an acid phosphatase leader (see, eg, WO90 / 13646). In mammalian cell systems, viral secretory leaders such as herpes simple gD signal and native immunoglobulin signal sequences (eg, human Ig heavy chains) are available. Typically, signal sequences are ligated in the reading frame with polynucleotides encoding the antibodies of the invention.

2.2 Replication Origin

Replication origins are widely known in the art as pBR322 suitable for most Gram-negative bacteria, 2μ plasmid for most yeasts, and various viral origins for most mammalian cells such as SV40, polyoma, adenovirus, VSV or BPV. Known. In general, the SV40 origin of the replication component is not essential to the mammalian expression vector, but SV40 origin may be included because it contains the initial promoter.

2.3 Selection Markers

Typical selection genes are: (a) tolerating antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, (b) supplementing nutritional deficiencies or providing nutrients not available in the complex medium ( c) encodes a protein that provides a combination of both. The selection scheme may include retarding the growth of the vector or host cell that does not contain the vectors. Cells successfully transformed with the gene encoding the therapeutic antibody of the invention survive, for example, due to drug resistance conferred by co-delivered selection markers. One embodiment is a DHFR selection system wherein the transformants are generated in a DHFR negative host strain (eg, see Page and Sydenham 1991 Biotechnology 9: 64-68). In this system, the DHFR gene is co-delivered with the antibody polynucleotide sequence of the present invention and then DHFR positive cells are selected by withdrawal. If necessary, DHFR inhibitor methotrexate is also applied to select DHFR gene amplified transformants. By operably binding the DHFR gene to the antibody coding sequence or functional derivative thereof of the present invention, DHFR gene amplification results in an additional amplification of the desired antibody sequence of interest. CHO cells are a particularly useful cell line for such DHFR / methotrexate selection and methods for amplifying and selecting host cells using the DHFR system are well established in the art. See, Kaufman R.J. et al J. Mol. Biol. (1982) 159, 601-621, for review, Werner RG, Noe W, Kopp K. Schluter M, "Appropriate mammalian expression systems for biopharmaceuticals", Arzneimittel-Forschung. 48 (8): 870-80, 1998 Aug.]. A further example is the glutamate synthase expression system (Bebbington et al Biotechnology 1992 Vol 10 p169). A suitable selection gene for use in yeast is the trp1 gene (see Stinchcomb et al Nature 282, 38, 1979).

2.4 promoter

Suitable promoters for expressing the antibodies of the invention are operably linked to DNA / polynucleotides encoding the antibodies. Promoters for prokaryotic hosts include phoA promoters, Beta-lactamases and lactose promoter systems, alkaline phosphatase, tryptophan and hybrid promoters such as Tac. Suitable promoters for expression in yeast cells include 3-phosphoglycerate kinases or other glycol enzymes such as enolase, glyceraldehyde 3 phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructo Kinases, glucose 6 phosphate isomerase, 3-phosphoglycerate mutases and glucokinase. Inducible yeast promoters include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein and enzymes that contribute to nitrogen metabolism or maltose / galactose use. Promoters for expression in mammalian cell lines include viral promoters such as polyomas, follox and adenoviruses (eg adenovirus 2), fetal papilloma virus, avian sarcoma virus, cytomegalovirus (especially immediate early). Gene promoter), retrovirus, hepatitis B virus, actin, Raus sarcoma virus (RSV) promoter and early or late Simian virus 40 and non-viral promoters such as EF-1alpha (Mizushima and Nagata Nucleic Acids Res 1990 18 (17): 5322), including an RNA polymerase II promoter. The choice of promoter is based on suitable compatibility with the host cell used for expression.

2.5 Enhancer Element

Suitably, for example, for expression in higher eukaryotes, additional enhancer elements may be included instead of those found to be located in the promoters described above. Suitable mammalian enhancer sequences include enhancer elements from globin, elastase, albumin, fetal protein, metallothionine, and insulin. Alternatively, enhancer elements from prokaryotic viruses such as the SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, baculovirus enhancer or murine IgG2a locus can be used (see WO04 / 009823). Such enhancers are typically located on vectors that are upstream to the promoter, but they may be located elsewhere, for example, in untranslated regions or downstream of the polyadenylation signal. Selection and positioning of the enhancer is based on suitable compatibility with the host cell used for expression.

2.6 Polyadenylation / termination

In the eukaryotic system, polyadenylation signals are operably linked to polynucleotides encoding the antibodies of the invention. This signal is typically located 3 'of the open reading frame. In mammalian systems, non-limiting examples include signals derived from growth hormone, kidney factor-1 alpha and viral (eg, SV40) genes or retroviral long term repeats. In yeast systems, non-limiting examples of polyadenylation / termination signals include those derived from phosphoglycerate kinase (PGK) and alcohol dehydrogenase 1 (ADH) genes. In prokaryotic systems, typically no polyadenylation signal is required, and it is common to apply shorter and more defined terminator sequences instead. The choice of polyadenylation / termination sequence is based on appropriate compatibility with the host cell used for expression.

2.7 yield Other ways to improve / element

In addition to the above, other features that may be applied to improve yield include chromatin remodeling elements, introns and host-cell specific codon modifications. Codon use of the antibodies of the invention can be modified to adjust the codon bias of the host cell to increase transcription and / or production yield (eg, Hoekema A et al Mol Cell Biol 1987 7 (8): 2914-24 ). The choice of codons is based on suitable compatibility with the host cell used for expression.

2.8 Host Cells

Suitable host cells for cloning or expressing a vector encoding an antibody of the invention include prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells are eubacteria (eubacteria), for example, enteric bacteria (enterobacteriaceae), for example, Escherichia (Escherichia), for example, Escherichia coli (E.Coli) (e. G., ATCC 31,446; 31,537; 27,325) , Enterobacter bakteo (Enterobacter), for El Winiah (Erwinia), keulrep when Ella Proteus (Klebsiella Proteus), for Salmonella (Salmonella) e.g., Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratia) e.g., Serratia dry process kanseu (Serratia marcescans) and Shigella (Shigella) and Bashile (Bacilli), for example, B. subtilis (B.subtilis) and B. Li Kenny formate miss (B.licheniformis) (see DD 266 710), Pseudomonas (Pseudomonas ) for example, to P. ah rugi it includes labor (P.aeruginosa) and Streptomyces (Streptomyces). Among yeast host cells, Saccharomyces cerevisiae , schizosaccharomyces pombe , Kluyveromyces (e.g. ATCC 16,045; 12,424; 24178; 56,500), The Yarrow (yarrowia) (EP 402,226), Pichia pastoris (Pichia pastoris) (EP 183,070, Document [Peng et al J.Biotechnol. 108 ( 2004) 185-192] reference), Candida (Candida), Trichoderma Silesia ( Trichoderma reesia) (EP244, 234) , penicillin (penicillin), toll-lipoic Cloud Stadium (Tolypocladium) and host, for example, Aspergillus (Aspergillus) nidul A. pullulans (A. nidulans) and A. nigeo (A.niger) is Is considered.

Although prokaryotic and yeast host cells are specifically contemplated by the present invention, typically, the host cells of the present invention are vertebrate cells. Suitable vertebrate host cells include mammalian cells such as COS-1 (ATCC No. CRL 1650) COS-7 (ATCC CRL 1651), human embryonic kidney 293, PerC6 (Crucell), baby hamster kidney cells (BHK) ( ATCC CRL.1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO.CRL 1573), Chinese hamster ovary cell CHO (eg, CHO-K1, ATCC NO: CCL 61, DHFR-CHO cell lines such as DG44 (see Urlaub et al, Somat Cell Mol Genet (1986) Vol 12, pp555-566)), especially CHO cell lines suitable for suspension culture, mouse sertoli cells, monkey kidney cells, African green monkey kidney cells (ATCC CRL-1587), HELA cells, dog kidney cells (ATCC CCL 34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or lymphoma cells eg NSO (see US 5,807,715), Sp2 / 0, YO have.

As such, in one embodiment of the present invention there is provided a stably transformed host cell comprising a vector encoding the heavy and / or light chain of a therapeutic antibody as described herein. Typically such host cells comprise a first vector encoding said light chain and a second vector encoding said heavy chain.

Such host cells may be further engineered or adjusted to modify the quality, function and / or yield of the antibodies of the invention. Non-limiting examples include the expression of specific modified (eg glycosylated) enzymes and protein folding chaperones.

2.9 Cell Culture

Host cells transformed with the vector encoding the therapeutic antibody of the invention can be cultured by methods known to those skilled in the art. Host cells can be cultured in spinner flasks, shake flasks, roller bottles, wave reactors (e.g., System 1000 from Wavebiotech Dot Com) or hollow fiber systems, and Production is preferred, and stirred tank reactors or bag reactors (eg, Wave Biotech, Somerset, New Jersey USA) are especially used for suspension culture. Typically, a stirred tanker is suitable for supplying air using, for example, a sparger, a baffle or a low shear impeller. In bubble columns and airlift reactors, direct air injection of air or oxygen bubbles may be used. When host cells are cultured in serum-free culture medium, a cell protective agent such as Pluronic F-68 is added to the medium to help prevent cell damage due to the air supply process. Depending on host cell characteristics, microcarriers can be used as growth substrates for adhesion dependent cell lines, or cells can be suitable for suspension culture (suspension culture is typical). Culture of host cells, particularly vertebrate host cells, can be carried out in a variety of modes of operation such as batch, fed-batch, repeated batch processes (see Drapeau et al (1994) cytotechnology 15: 103-109), extended batch processes. Or perfusion culture can be used. Recombinantly transformed mammalian host cells can be cultured in serum containing media such as fetal bovine serum (FCS), but such host cells can be replaced with energy sources such as glucose and synthetic growth factors such as Serum-free media or commercially available media, such as ProCHO-CDM or UltraCHO ^™ (Cambrex NJ, supplemented with recombinant insulin, such as described in Keen et al (1995) Cytotechnology 17: 153-163) Cultivated in USA). Serum-free culture of host cells should be suitable for these cells to grow in serum-free conditions. One adaptation method is to cultivate such host cells in serum-containing medium repeatedly replacing 80% of the culture medium for serum-free medium to adapt the host cells to serum-free conditions (Scharfenberg K et al ( 1995) in Animal Cell technology: Developments towards the 21st century (Beuvery EC et al eds), pp619-623, Kluwer Academic publishers.

Antibodies of the invention secreted into the medium are recovered and purified from the medium using a variety of techniques to provide a degree of purification suitable for the intended use. For example, the use of a therapeutic antibody of the invention for the treatment of a human patient is typically at least 95% pure, as measured by reducing SDS-PAGE, as compared to the culture medium comprising the therapeutic antibody, More typically it should be 98% or 99% pure. In a first example, cell debris from the culture medium is typically removed by centrifugation followed by a supernatant clarification step using, for example, microfiltration, ultrafiltration and / or depth filtration. Alternatively, the antibodies can be recovered by microfiltration, ultrafiltration or deep filtration without prior centrifugation. Various other techniques such as dialysis and gel electrophoresis and chromatography techniques such as hydroxyapatite (HA), affinity chromatography (optionally including an affinity tagging system such as polyhistidine) and / or hydrophobic interaction chromatography (See HIC, US 5,429,746). In one embodiment, after various purification steps, the antibody of the invention is subjected to protein A or G affinity chromatography followed by further chromatography steps such as ion exchange and / or HA chromatography, anion or cation exchange, size exclusion chromatography. And ammonium sulfate precipitation. Typically, various virus removal steps are also used (eg, nanofiltration using a DV-20 filter). After these various steps, a purified (preferably monoclonal) preparation is provided comprising at least 10 mg / ml or more, for example 100 mg / ml or more of the antibody of the invention. Embodiments of the invention are formed. Ultracentrifugation can produce concentrations of 100 mg / ml or more. Suitably such preparations are virtually free of antibodies in the aggregated form of the invention.

Bacterial systems are particularly suitable for expressing antibody fragments. Such fragments are localized within the cell or in periplasma. Soluble periplasmic proteins can be extracted and refolded to form active proteins according to methods known to those of skill in the art. See Sanchez et al (1999) J. Biotechnol. 72, 13-20 and Cupit PM et al (1999) Lett Appl Microbiol, 29, 273-277.

3. Pharmaceutical Composition

Purified preparations of the antibodies of the invention as described above (particularly monoclonal preparations) may be incorporated into pharmaceutical compositions for use in the treatment of human diseases and conditions such as those listed above. Typically, such compositions further comprise a pharmaceutically acceptable (ie inert) carrier as known and required for acceptable pharmaceutical practice (eg, Remingtons Pharmaceutical Sciences, 16th edition). , (1980), Mack Publishing Co). Examples of such carriers are sterile carriers, such as saline, Ringer's solution, or dextrose solution, which have been buffered to a pharmaceutically acceptable pH such as pH 5 to 8 by suitable buffers such as sodium acetate trihydrate. Pharmaceutical compositions for infusion (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular or intraportal) or for continuous infusion are preferably free of visible particulate material and for the treatment of 1 mg to 10 g Antibodies, typically 5 mg to 1 g, more particularly 5 mg to 25 mg or 50 mg of antibody. Methods of preparing such pharmaceutical compositions are well known to those skilled in the art. In one embodiment, the pharmaceutical composition comprises 1 mg to 10 g of the therapeutic antibody of the invention, optionally in unit dosage form with instructions for use. The pharmaceutical compositions of the present invention may be lyophilized (freeze dried) and reconstituted prior to administration according to methods well known or apparent to those skilled in the art. When an embodiment of the invention comprises an antibody of the invention having an IgG1 isotype, a chelator of metal ions comprising copper, such as citrate (eg sodium citrate) or EDTA or histidine, is incorporated into the pharmaceutical composition. It can be added to lower the metal-mediated degradation level of this isotype antibody (see EP 0612251). The pharmaceutical compositions may also include solubilizers such as arginine bases, detergents / anti-agglomerates such as polysorbate 80, and inert gases such as nitrogen to replace the space-part oxygen in vials.

Effective doses and treatment regimens for administering the antibodies of the invention are generally determined experimentally and depend on factors such as the age, weight and health of the patient, and the disease or condition to be treated. These factors are within the authority of the primary care physician. Criteria for selecting a suitable dosage are found, for example, in Smith et al (1977) Antibodies in human diagnosis and therapy, Raven Press, New York, and will generally be from 1 mg to 10 g. In one embodiment, the dosage regimen for treating a human patient is 1 mg to 1 g of the therapeutic antibody of the invention, administered subcutaneously once a week or once every two weeks, or intravenously injected every month or two months. . This dosage corresponds to 0.014-140 mg / kg, such as 0.014-14 mg / kg. The composition of the present invention may be used for prevention.

4. Clinical Uses

Diseases characterized by increased β-amyloid levels or β-amyloid deposition include Alzheimer's disease, mild cognitive impairment, Down's syndrome, hereditary cerebral hemorrhage with Dutch type β-amyloidosis, cerebral β-amyloid angiopathy, and various types of degenerative dementia Such as those associated with Parkinson's disease, progressive nuclear paralysis, cortical key degeneration and diffuse Alzheimer's disease.

Most preferably, the disease is Alzheimer's disease characterized by increased β-amyloid levels or β-amyloid deposition.

Although the present invention has been described primarily with regard to the treatment of human diseases or conditions, the present invention can also be applied to the treatment of similar diseases or conditions in mammals other than humans.

Way

A device for measuring real-time kinetics of molecular interactions using the Biacore ™ / Biacore 3000 SPR

Surface Plasmon Resonance (SPR)-a physical phenomenon applied by the Biacore ™ machine to measure mass changes on sensor chips

Biacore ™ sensor chip with general purpose surface coated with CM5 carboxymethylated dextran matrix

ELISA enzyme immunoassay

SRU BIND ™ biosensor technology to monitor SRU-labeled biochemical interactions

Mini-Bioreactors sold by Integra CL 1000 IBS Integra Biosciences

FMAT fluorescence microvolume assay technology (Applied Biosystems)

Applied Biosystems 8200 Fluorescence Macro Confocal Cell Detection System for ABi8200 FMAT

FPLC High Speed Protein Liquid Chromatography

Protein A column for FPLC sold by ProSepA HiTrap GE Healthcare

material

DMSO Dimethylsulfoxide

HEPES N- (2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid)

EDTA ethylenediaminetetraacetic acid

Tris HCl-Tris- (hydroxymethyl) aminomethane hydrochloride

NaCl-Sodium Chloride

Tween-20-polyoxyethylene sorbitan monolaurate

BSA-bovine serum albumin

PBS-phosphate buffered saline

PFA-Paraformaldehyde

IMS-Industrial Methylated Spirit

DAB-3,3'diaminobenzidine

DMEM Dulbecco's Modified Eagle Medium

FCS Fetal Bovine Serum

Medium Based on Modified Eagle Medium by Opti-MEM Invitrogen / Gibco

Cationic Lipid Based Cell Transfection Agents Sold by Lipofectamine Invitrogen / Gibco

Liposome transfection agents sold by Transfast Promega

Versene metal ion chelating agent (ethylenediaminetetraacetic acid)

Stable form of glutamine (dipeptide L-alanyl-L-glutamine supplement) added to Glutamax culture medium

Histoclear tissue cleaner

HBS-EP Buffer General purpose Biacore ™ buffer containing 0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20.

Generation of Mouse Monoclonal Antibody 2E7

Mouse monoclonal antibody 2E7 was generated from normal immunization of mice. Mice were immunized with soluble or aggregated β-amyloids 1-40 and 1-42 formulated in Freund's adjuvant. After the final boost without adjuvant, splenocytes were fused with myeloma cells. Fusion cells were grown in 96-well plates, from which hybridoma supernatants were screened for potential leads. Selected antibody 2E7 obtained by immunization with soluble β-amyloid 1-40 was a murine lgG2a isotype and had beta-amyloid binding activity in the effluent assay described below and affinity was determined by Biacore ™, Method A (i). 36.1 pM for amyloid 1-40 (Table 10A).

Epitope Mapping in 2E7

To precisely map the binding of antibody 2E7 to the β-amyloid peptide, peptide set (A) was used. Peptide set (A) consists of a set of 31 12-mer overlapping peptides covering the complete sequence of β-amyloid 1-42 peptide. Each contiguous peptide is initiated from contiguous amino acids in the β-amyloid peptide, thus changing the sequence covered between contiguous peptides by a single amino acid. All peptides in set (A) contained three amino acid C-terminal linkers (glycine-serine-glycine) and terminal biotinylated lysine residues. In addition, all peptides except for peptide Aβ1 DAEFRHDSGYEVGSGK-Biotin (SEQ ID NO: 15) were acetylated at the N-terminus. The second set of peptides (set (B)) consists of one contiguous amino acid C-terminal deletion from a peptide containing amino acids 1-10 of the β-amyloid sequence. All peptides in set (B) contained three amino acid C-terminal linkers (glycine-serine-glycine) and terminal biotinylated lysine residues, but replaced additional glycine and serine residues in place of the deleted β-amyloid amino acid. (Table 2). Thus, all peptides in set (B) have the same length.

Table 2

Sequence of biotinylated peptide containing truncated N-terminal fragment of β-amyloid (set (B))

Binding Monitoring of β-amyloid-derived Peptides of 2E7 Using an Optical Biosensor

96-well SRU Bind ™ streptavidin-coated plates (SRU Biosystems) were washed with PBS containing 1% DMSO to remove glycerol and preservatives. A constant baseline was provided by leaving a volume of 50 μl / well to equilibrate at room temperature. Biotinylated peptide was diluted to approximately 0.3 μg / ml in PBS containing 1% DMSO and 50 μl each was added to the wells and incubated for about 1 hour. Duplicate wells were prepared using different sectors of the plate and at least one non-peptide control well was used as a reference to subtract data from each sector. After peptide capture, the plates were washed with PBS containing 1% DMSO and 50 μl fresh buffer was left per well to provide a new baseline on the reader. No disruption of the peptide was observed from the surface. The buffer was then replaced for 2 hours with 40 μl / well buffer containing 20-64 nM test antibody. It was found that antibody 2E7 bound only to peptides (peptide Aβ1, SEQ ID NO: 15) comprising amino acids 1-12 of the β-amyloid peptide in peptide set (A). This result suggests that aspartic acid of residue 1 is required for binding to this peptide.

To further characterize the binding site of antibody 2E7, peptide set (B) was used. Using the SRU BIND ™ biosensor method, antibody 2E7 showed negligible binding to peptides (SEQ ID NOS: 14 and 13) comprising amino acids 1-3 and 1-4 of the β-amyloid peptide. The binding of the β-amyloid peptide to the peptide comprising amino acids 1-7 (SEQ ID NO: 10) was comparable to the peptide (from peptide set (A)) comprising amino acids 1-12 of the β-amyloid peptide. Binding of the β-amyloid peptide to peptides comprising amino acids 1-5 or 1-6 (SEQ ID NOs 12 or 11) was observed, but to peptides comprising amino acids 1-7 of β-amyloid peptide (SEQ ID NO: 10) It was weaker than binding for (measured by stability after additional wash steps).

Thus, only residues 1-7 of the β-amyloid peptide were found to be necessary for sufficient binding when measured using this method.

Surface plasmon resonance black

In addition to the experiments described above, binding of 2E7 antibodies to selected β-amyloid sequence derived peptides was monitored using a Biacore ™ 3000 optical biosensor. Binding was measured by injecting test antibodies onto the peptide captured on the streptavidin chip surface (130-230 RU (Resonance Unit)) separated for 5 minutes below 64 nM. Running buffer (HBS-EP) containing 0.01 M HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA and 0.005% surfactant P20 ™ was used at a flow rate of 20 μl / min at 25 ° C. All progress was assessed in duplicate for blank streptavidin surface and blank injection. Analysis was performed using Biacore ™ analysis software BIAevaluation ™ version 4.1. Results from selected peptides of set (A) show data derived from SRU BIND ™ indicating that 2E7 binds only to a peptide comprising amino acids 1-12 of β-amyloid peptide (SEQ ID NO: 15) with an apparent equilibrium constant KD of about 50 Further confirmation. Under the same conditions, 2E7 did not bind to the peptide comprising amino acids 2-13 of the β-amyloid peptide.

Peptide Aβ2-13 AEFRHDSGYEVHGSGK-Biotin (SEQ ID NO: 44)

The experimental methods and conditions used allow detection of not only high affinity molecules but also low affinity molecules—in the same experimental setup, in contrast to 2E7, another antibody that recognizes the N-terminal epitope of β-amyloid peptide An apparent KD of 7 nM was shown to bind to 2-13 peptide (SEQ ID NO: 44). 2E7 did not bind to the selected peptide of set (A) from the middle region of the β-amyloid peptide. In a separate experiment, β-amyloid 1-40 peptide was captured via its biotinylated N-terminal aspartic acid residue. This peptide was captured on the Biacore ™ streptavidin coated chip described above. Antibody 2E7 injected at 66 nM for 1 minute could not bind this peptide. Thus, it was concluded that the N-terminal binding site described above is masked by the linker and capture method, thus confirming that the N-terminal terminal contains the core binding site.

Binding to Cell Expressed Amyloid Precursor Protein (APP)

β-amyloid consists of a peptide formed by proteolytic cleavage of a type I transmembrane precursor protein called amyloid precursor protein (APP). Because APP is a large extracellular domain, binding to this protein could potentially initiate an antibody-dependent cellular cytotoxic response (ADCC).

The FMAT ™ ABI8200 based assay was used to characterize the binding of antibodies to cell-surface full length APP.

Transfection of HEK293T Cells Using Wild-Type APP DNA

HEK293T cells are maintained in DMEM F12 medium containing 10% (v / v) FCS and 1 × Glutamax. Cells were seeded in 75 cm ² tissue culture flasks and grown to 60-80% confluence (18-24 h) prior to transfection. For transfection, 9 μg of DNA (wild type APP DNA (in PCDNA3.1 (Invitrogen) vector), or control of the vector alone) was mixed with 0.3 ml of Opti-MEM ™ medium. 30 μL of Lipofectamine ™ Transfection Agent was mixed with 0.3 ml Opti-MEM ™ medium and both mixtures pooled. The pooled mixture was incubated at room temperature for 30 minutes before additional 4.8 ml of Opti-MEM ™ medium was added. The final mixture was added to the cells (after washing with Opti-MEM ™ medium) for 5 hours and then 6 ml of 10% (v / v) newborn calf serum in DMEM. After 48 hours of transfection, the supernatant was removed and the monolayer was washed with Versen, then 3 ml of Versen ™ chelating agent was added to each flask, incubated at 37 ° C. for 5 minutes, and 200 g of the isolated cells were Pelleted for 5 minutes. The resulting cell pellet was slowly resuspended in 1 ml of assay buffer (2% heat treated serum in PBS, 0.5% BSA, 0.1% NaN ₃ pH7.4, filtered through 0.2 μm filter) to produce a single cell suspension.

FMAT ™ ABI8200 Substrate Assay

Assay buffer sterile filtered in polypropylene plates with test antibody (2E7, LN27 (Zymed) mouse IgG for extracellular domain of APP as positive control, and antibody G210 that recognizes x-40 form of β-amyloid as negative control) (2% heat treated serum in PBS, 0.5% BSA, 0.1% NaN ₃ pH7.2) to 10 μg / ml, followed by an additional six successive 1: 1 dilutions along the plate. Only assay buffer was used as blank. 50 μl suspension of HEK293T cells transfected with wild-type APP DNA (Experiment 1: 10,000 cells; Experiment 2: 20,000 cells) was added to each well of a 96 well plate, and 5 μl of each antibody solution was added thereto. The wells were doubled by addition. 50 μl / well of F-MAT ™ blue anti-mouse IgG stock (antibodies were F-MAT ™ blue work from ABI) diluted 1: 500 (Experiment 1) and 1: 1000 (Experiment 2) in assay buffer Labeled using the Sex Reaction Dye Kit, 4328408) was added to each well and the plate was shaken briefly and allowed to settle for 1 hour. Plates were then read using ABI 8200 fluorescent macro confocal cell detection system (Applied Biosystems).

The acquired aggregated data was interpreted using Excel ™ spreadsheet software. In brief, mock transfected aggregates were subtracted from full length APP transfected cell aggregates to obtain specific signals for each antibody. Two antibody concentrations in the straight portion of the curve were selected (1.25 and 0.63 μg / ml) and the background corrected obtained aggregates at these concentrations were expressed as a percentage of LN27 antibody binding and averaged over both antibody concentrations. The data obtained are described in Table 3 (% ± SE of LN27 binding). Thus, within this assay system, the binding of 2E7 to cell surface APP cannot be distinguished from that of negative control antibody G210.

TABLE 3

Binding to Amyloid Precursor Protein-Derived Peptides

The epitope mapping studies disclosed above showed that antibody 2E7 is bound to the N-terminus of the terminal of the β-amyloid peptide, and that the aspartic acid residue at position 1 is essential for binding. This suggests that the antibody recognizes a 'neo' epitope formed by cleavage of APP at the β-secretase site. This observation would suggest that antibody 2E7 should not recognize adjacent APP peptide sequences. To test this hypothesis, an APP peptide (peptide APP1, SEQ ID NO: 16) comprising residues 1-7 and 5 contiguous APP derived amino acids of the β-amyloid peptide was synthesized. Thus, peptide APP1 contained consecutive amino acids from the position 5N-terminus for the BACE-1 cleavage site to the position 7 C-terminus for the BACE-1 cleavage site and was N-terminally acetylated. The ability of antibody 2E7 to bind APP derived peptides APP1 and β-amyloid 1-12 peptide (peptide Aβ1) was compared using the Biacore ™ method (described previously for epitope mapping). Antibody 2E7 showed high affinity binding to β-amyloid peptide Aβ-1 containing basic epitopes 1-7. However, no binding was observed for the APP1 peptide, which also contained the basic β-amyloid derived sequences 1-7.

Peptide Aβ1 DAEFRHDSGYEVGSGK-Biotin SEQ ID NO: 15

APP1 AcNH-SEVKMDAEFRHDGSGK-Biotin SEQ ID NO: 16

Using a combination of FMAT ™ based cell binding and Biacore ™ based peptide mapping, in this format, 2E7 had no binding affinity for the full length APP protein. Assuming that aspartic acid residues at position 1 of the β-amyloid peptide are required for binding, it is concluded that only 2E7 recognizes the 'neo' N-terminus of β-amyloid and therefore should not bind to cell surface expressed APP.

Biological activity in vivo

I ¹²⁵  β-amyloid spill model

Many published studies have shown that β-amyloid antibodies can complex with β-amyloid peptides in the bloodstream. It is claimed that this sequestration of peripheral β-amyloid enables further outflow of CNS amyloid into the bloodstream (DeMattos RB, PNAS (2001), 98 (15); 8850-8855). Acute pharmacodynamic models have been developed to screen antibodies for their ability to complex with brain-derived β-amyloid peptides in the bloodstream.

Anesthesia (4% isoflurane) of male C57 / BL6J mice was induced and maintained in 100% oxygen (1.5% isoflurane). The animal was then placed in the stereotactic frame. After midline incision following sagittal suture, a bore hole was drilled through the skull and a guide cannula was inserted into the lateral ventricle (co-vertical anterior and posterior (AP) -0.5 mm, lateral (L) +0.7 mm). , Ventral (V) -2.5mm). In addition, two bore holes were drilled through the skull and the cortical screw was positioned thereon. The cannula was anchored in place by a cyanoacrylate gel and the incision was closed around the cyanoacrylate gel headcap. After surgery, mice were given subcutaneous 0.3 ml saline and placed in a warm environment to recover from anesthesia. Upon recovery of corrective reflexes, mice were housed alone and received standard post-operative care for 5 days. No action was taken for an additional 5 days or until preoperative weight was restored. After recovery, the cannula position was confirmed by angiotensin II drinking reaction. Each mouse received 100 ng of angiotensin II (AII) (prepared in 0.9% saline) intraventricularly (ICV). After administering AII, water intake was observed for 15 minutes. Mice with a sustained positive response to AII (sustained drinking) were included in the study, which was initiated 5 days after AII infusion.

On the day of the study, mice were placed in a warm environment for 5-10 minutes to induce vasodilation, which is necessary to facilitate injection into the tail vein. Test antibodies (600 μg) or PBS vehicles (dose volumes up to 10 ml for 1 kg body weight) were injected through the tail vein and returned to their individual cages after injection. Exactly 1 hour after injection into the tail vein, mice were slowly ICV injected (2 μl per minute) of 2 ng (1 μCi) of I ¹²⁵ beta-amyloid 1-40 (Amersham Biosciences, UK) at a dose volume of 5 μl. Exactly 4 hours after ICV administration, 50 μl of arterial blood was collected and radioactivity measured on a scintillation counter.

Mice injected with 2E7 (n = 6 per treatment group) in the tail-vein had a statistically significant increase in radioactivity signal (coefficient-CPM) in 50 μl of arterial blood compared to the CPM signal detected in vehicle-injected mice. -(CPM-vehicle: 1339.7 ± 496.2 vs. 2E7 4387.9 ± 980.3; ANOVA: F (2,13) = 4.97, p <0.05.Post-hoc LSD: p = 0.01 2E7 vs. vehicle [post-hoc Duncans: p = 0.02 2E7 vs, vehicle]).

In two further studies with 2E7 performed using the same protocol, a similar increase in amyloid efflux into the blood was observed compared to vehicle injected controls (CPM blood: vehicle 352 ± 113 versus 2E7 2397 ± 353, and vehicle 1281 ± 312 versus 2E7 5291 ± 885; p <0.001 vs. vehicle using ANOVA post-hoc LSD test).

Transgenic CNS β-amyloid Degradation Model

1. β-amyloid load after 4 weeks of administration of 2 month-old TASTPM mice

Male and female TASTPM transgenic mice (dual-mutated APPswe x PS1.M146V, Howlett DR (2004) Brain Research 1017 (1-2) 130-136) at the start of the study were 61-65 days old and received alone. The same number of mice were assigned to each treatment group (n = 12 per group) and randomized according to gender and age. Treatment groups included: A: MOPC21 (antibody of unknown specificity, Holton et al (1987) J. Immunol 139 (9) 3041-3049, negative control), B: 2E7 (test antibody). All antibodies were dissolved in PBS and administered by the intraperitoneal route. Regardless of the weight of the animal, 300 μg of antibody was administered. Animals were administered twice weekly for four weeks. One day after the last dose, animals were euthanized with excess sodium pentobarbital. The brain was dissected and cut in half. Half-cut brain samples were collected in pre-weighed 2 ml Eppendorf ™ tubes and snap frozen. Subsequently, the samples were thawed, reweighed, and 1 ml of 5M guanidine HCl containing complete Protease Inhibitor ™ tablets (Boefringer Mannheim) was added, then the samples were homogenized and incubated for> 90 minutes at 4 ° C. with constant stirring. .

Samples are then diluted to 1 in 10 in assay buffer (50 mM Tris HCl, pH7.4, 150 mM NaCl, 0.05% Tween-20 + 1% BSA), vortexed, and spun at 20 ° C. for 20 minutes at 20 ° C. I was. The supernatant was removed and added to the assay plate as a triple sample.

C-terminal specific β-labeled with an Oritag ™ specific label to facilitate detection (BioVeris ™) used to capture Aβ40 or Aβ42, with biotinylated N-terminal specific Aβ antibodies A β40 and Aβ42 levels were measured using a sensitive plate based electrochemiluminescent immunoassay (BioVeris ™) applying an amyloid antibody (for Aβ40 or Aβ42). Antibody-Aβ complexes were captured with streptavidin coated beads bound to biotinylated antibody (Dynabeads ™, Dynal) incubated overnight at room temperature with intense mixing and assayed in a BioVeris ™ M8 photodetector. Standard curves were drawn using human Aβ40 and Aβ42 peptides in assay buffer containing the required concentration of guanidine HCl. Data was analyzed using Excel Robosage ™ statistical analysis software and Aβ levels were expressed as pmole / tissue g.

In this legend, treatment with 2E7 antibody reduced CNS Aβ42 load by 37% (p <0.001) and CNS Aβ40 load by 23% (p <0.001).

In subsequent studies under similar experimental conditions, 2E7 antibodies compared CBS Aβ42 load with 38% (Study 1, males only), 22% (Study 2, non-significant) and 39% (Study 3, Males, p = 0.001) and 13% (study 3, females, not significant). In these studies, 2E7 also reduced CNS Aβ40 by 18% (Study 3, male, p = 0.017) compared to PBS treated animals and 25% (Study 1, male only), <1% in CNS Aβ40 (Study 2). ), And a significant increase of 3% (Study 3, female).

2. β-amyloid load after 4 months of administration of April-old TASTPM mice

In brief, four-month-old TASTPM transgenic mice were administered 300 μg of antibody once or twice weekly via the intraperitoneal (i.p.) route. After 4 months of administration, CNS β-amyloid levels were measured by ELISA and plaque load was measured by immunohistochemistry. Between April and August, CNS β-amyloid loads increased dramatically, resulting in plaque pathology rapidly developing (Howlett DR (2004) Brain Research 1017 (1-2) 130-136).

Mice were 120-128 days old at the start of the study and received alone. Similar numbers of mice were assigned to each treatment group (n = 20 or 21 per group) and randomized by gender and age. Treatment groups included: A: PBS administered twice weekly (vehicle), B: 2E7 administered once weekly, C: 2E7 administered twice weekly, D: PBS administered once weekly. 300 μg dose (79 μl volume) of 2E7 was administered via the intraperitoneal route. Vehicle treated animals received the same volume of PBS. Animals were administered for 18 weeks. TASTPM mice are susceptible to spontaneous seizures, resulting in the death of many animals during the study. Final numbers were as follows: A: 4 females, 9 males; B: 5 females, 8 males; C: 4 females, 9 males; D: 2 females, 9 males. Two or four days after the last dose (same number per group) animals were euthanized with excess sodium pentobarbital. Tail tip samples were taken from each mouse to confirm genotype. The brain was dissected and cut in half. The right hemispheres were fixed by immersion in 4% paraformaldehyde and processed for histology. The left hemispheres were collected in pre-weighed 2 ml Eppendorf ™ tubes, frozen on dry ice and stored at −80 ° C. for subsequent analysis of amyloid content. Prior to analysis, samples are thawed, reweighed, and 1 ml of 5M guanidine HCl containing complete Protease Inhibitor ™ tablets (Boefringer Mannheim) is added, then the samples are homogenized and incubated for> 90 minutes at 4 ° C. with constant stirring. It was.

Samples are then diluted in assay buffer to 1 in 10 (50 mM Tris HCl, pH7.4, 150 mM NaCl, 0.05% Tween-20 + 1% BSA), vortexed, and spun at 20 ° C. for 20 minutes at 20 ° C. I was. The supernatant was further diluted 1: 1000 and added to the assay plate as a triple sample. Levels of Aβ40 and Aβ42 were measured as a four week dosing study.

Variance analysis was used for treatment, gender and dosing schedules included in the model as fixed effects. All interactions between the three factors were included. There was no significant difference between the two dosing schedules (once or twice weekly). In this experimental design, it was first possible to assess whether there was any significant difference between the dosing schedules, and secondly, since this significance did not exist between the two dosing schedules, the data from them could be combined, so By doubling the number of mice, the power of the experiment could be increased.

In this legend, treatment with 2E7 antibody reduced the CNS Aβ42 load by 22.5% (p = 0.0152). The level of CNS Aβ40 was also reduced by 12.1%, but this level did not reach statistical significance (p = 0.118).

Complex immunohistochemical analyzes of these samples were performed to define the brain tissue area indicative of plaque pathology. Sections were taken from the tail level cortex and the hippocampus level cortex. Adjacent sections were stained with Aβ40 or Aβ42 specific antibodies or alternatively amyloid stained Congo Red. Using image analysis software, the area of the stained sections for plaques was expressed as a percentage of the total section area.

After fixation, PFA-soaked half brains were cut coronally into sections 6 × 2 mm thick in the brain matrix. This 2 mm section is referred to as sections A through F, where A is the mouth and F is the tail. Sections A, B & C and D, E & F were placed in separate embedding cassettes numbered for each animal. The cassette was kept in PFA until processing and embedding preparation.

Embedding was performed on a Citadel ™ 1000 (Shandon) tissue processor. All tissues followed the following processing regimens:-

70% IMS-1 hr

100% IMS-3 x 1 hr

100% Ethanol-2hr

100% isobutyl alcohol; 1 x 2hr; 1 x 1.5hr

Histoclear ™-2 x 1.5hr

Paraffin wax-2 x 2hr

After completing the processing cycle, the wax saturated tissue sections were transferred to a molten-paraffin wax filled base mold and embedded using a Histocentre ™ (Shandon) paraffin embedding system. Sections A, B & C go into one mold; The tissue was embedded so that D, E & F went to the second mold. This was done for all sets of sections, ie each half-cut brain, resulting in two wax blocks of three sections each. Sections were placed in the mold so that the tail surface of each piece was the future cut surface. Care was taken to ensure that each section was pushed down well in the mold so that each microtoming occurred at the same time. The perforated processing cassette was carefully placed on each mold and covered with molten wax thereon. The embedded blocks were cooled on cooled plates until they could be removed from the mold. Blocks were stored at room temperature until needed for microtoming. Blocks were optionally cut and 5 micron sections were suspended in pre-labeled gelatin coated slides (Superfrost ™, Erie Scientific Company). Two sections were suspended on each slide. Wherever possible, consecutive sections were mounted and slides were numbered consecutively from 1 to 25. 50 sections (25 slides) were taken from each block. The slides were dried on hot plates and then stored at room temperature until needed.

Immunohistochemistry was performed on 30 slide sets. For each slide, the upper section is labeled with Aβ40 antibody (G30, rabbit polyclonal that recognizes x-40 β-amyloid) and the lower section is monoclonal that recognizes Aβ42 antibody, 20G10, x-42 β-amyloid Labeled with raw antibody. At least 5 sections per antibody were labeled for the block.

Labeling was performed as follows. Sections were waxed with histoclear and graded alcohol, then immersed in 85% formic acid for 8 minutes and blocked with 0.3% hydrogen peroxide for 30 minutes to block endogenous peroxidase. Both antibodies G30 and 20G10 were applied overnight at 1: 1000 dilution and the sections were kept at 4 ° C. The development of the sections was made with each biotinylated anti-rabbit and anti-mouse secondary. Color development was achieved using a diaminobenzidine tetrahydrochloride staining kit (DAB ™, Vector Labs). Sections were simply counterstained with Mayer's hematoxylin, then dehydrated, cleaned, and glass-covered.

Sections were left to dry for at least 48 hours before microscopy. Images were captured on a Leica DMRB ™ microscope equipped with a digital camera. Images were analyzed using Qwin ™ software (Leica) and the results were expressed as% of cross-sectional area labeled with Αβι_ι antibodies.

Variance analysis was used for treatment, gender and dosing schedules included in the model as invariant effects. All interactions between the three factors were included. There was no significant difference between the two dosing schedules (once or twice weekly). In this experimental design, it was first possible to assess whether there was any significant difference between the dosing schedules, and secondly, since this significance did not exist between the two dosing schedules, the data from them could be combined, so By doubling the number of mice, the power of the experiment could be increased.

In this legend, treatment with 2E7 antibodies reduced plaque pathology as measured by antibodies that recognize Aβ42. Plaque pathology was reduced by 27.1% (p = 0.0026) in the cortex at the hippocampus level and by 43% (p <0.0001) in the cortex at the tail level. Plaque pathology was also reduced when measured with an antibody that recognizes Aβ40. Plaque pathology was reduced by 16.6% (p = 0.0421) in the cortex at the hippocampus level and by 17.3% (p = 0.0342) in the cortex at the tail level.

No evidence of microbleeding was observed in any mice from this study treated with vehicle or 2E7 (measured by Perls' Prussian Blue). This method visualizes ferric iron (iron is an essential component of oxygen-carrying hemoglobin found in red blood cells) by producing soluble blue compounds. All brain levels from all animals were clean.

Cognitive model

After 4 months of administration to 4 month old TASTPM mice as described above, these mice were tested in two cognitive models: object recognition assay and fear control assay.

Object recognition test

Object recognition tests use the innate tendency of animals to explore new objects and rely on the animal's ability to recall previously explored objects (used objects). August-old TASTPM mice have been shown to lack the ability to distinguish between new and familiar objects (Howlett et al., 2004) and this indicates impaired recognition performance in these animals. However, in this study, vehicle-treated 8-month-old TASTPM mice did not demonstrate impaired cognition, ie they could distinguish between new and familiar objects. Thus, there was no means to investigate any potential therapeutic effects from treatment with 2E7.

Fear control black

A fear control model was designed to test the animal's ability to recall previous painful stimuli with situations or signaled signals when the same situation or tone was provided after the Xh delay. In this study, vehicle-treated (once or twice weekly) 8-month-old TASTPM mice showed a deficit in the contextual labeling of cognitive impairment in these animals. This deficiency was not affected by treatment with 2E7 once or twice weekly.

4-month administration of 6-month-old TASTPM mice

This study included administration of 2E7 (300 μg i.p. twice weekly) for 4 months in TASTPM mice, starting at March age. Control animals received IgG2A in PBS. As described above, the brain was dissected and cut in half. The right hemispheres were fixed by immersion in 4% paraformaldehyde and processed for histology. The left hemispheres were collected in pre-weighed 2 ml Eppendorf ™ tubes, frozen on dry ice and stored at −80 ° C. for subsequent analysis of amyloid content.

Preliminary analysis of a single section by IHC from each randomly selected brain sample (n = 6 vehicle, n = 7 2E7 treatment group) was performed using the same general protocol as above. Statistical analysis (Student t-test) showed a significant decrease in Aβ42 plaque load in thalamus (71.9%, p = 0.007) and thalamus + cortex + hippocampus (54.1%, p = 0.022) in mice dosed with 2E7 and at Aβ40 There was no significant change.

For biochemical measurements of brain Aβ40 and Aβ42, samples were processed and measured as above (dilution factor 1: 10,000). Aβ 42 was significantly reduced by 29.9% in mice dosed with 2E7 (p = 0.01) (n = 12 control, n = 16 treated). Aβ40 concentrations also decreased (22.6%), but this reduction did not reach statistical significance (p = 0.052).

Cloning of Hybridoma Variable Regions

Variable region sequence

Total RNA was extracted from 2E7 hybridoma cells and heavy and light chain variable domain cDNA sequences were generated by reverse transcription and polymerase chain reaction (RT-PCR). The forward primer for RT-PCR was a mixture of degenerating primers specific for the murine immunoglobulin gene leader-sequence and the reverse primer was specific for the antibody constant region, in this case murine isotype IgG2a for heavy chain and murine kappa for light chain . Primers were designed according to the method described in Jones and Bendig (Bio / Technology 9:88, 1991). RT-PCR was performed in duplicate for both V-region sequences allowing for subsequent verification of the correct V-region sequences. The V-region product generated by RT-PCR was cloned (Invitrogen TA Cloning Kit) and sequence data was obtained.

2E7 V _H amino acid sequence (SEQ ID NO: 17)

2E7 V _H DNA Sequence (SEQ ID NO: 18)

2E7 V _L amino acid sequence (SEQ ID NO: 19)

2E7 V _L DNA Sequence (SEQ ID NO: 20)

Complementarity determining regions (CDRs) are underlined in the amino acid sequence.

Cloning and Expression of 2E7 Chimeras

Accurate cloning of the functional murine V region was generated by generating a chimeric 2E7 antibody (2E7c) consisting of a parental murine V region transplanted onto human IgG1 (Fc mutated (L235A, G237A)) for the heavy chain or a human C kappa region for the light chain. Recombinant antibody material was expressed that could be used to confirm. Mammalian expression vector RLD-bshe (on the heavy chain) already containing the human constant region (eg IgG1 Fc mutated (L235A, G237A) or human C kappa) of the DNA encoding the 2E7 murine heavy and light chain V regions and the endogenous murine signal sequence ) And RLN-bshe (for light chain) in the frame.

Elements of the RLD-bshe expression vector for heavy chain expression:

Description of Base Pair DNA Segments

0-1014 promoter (SV40 / RSV)

1015-2442 Antibody Heavy Chain

2443-2765 Poly A

2766-3142 BG promoter

3239-3802 DHFR

3803-4162 Poly A

4163-6322 total backbone

5077-5937 (complementary strand) beta lactamase

(The location of the provided elements and the overall size of the vector are for illustrative purposes only and will depend on the size of the antibody chain insert)

Elements of the RLN-bshe expression vector for light chain expression:

Description of Base Pair DNA Segments

0-1014 promoter (SV40 / RSV)

1015-1731 Antibody Light Chain

1732-2051 Poly A

2388-2764 BG promoter

2774-3568 Neomycin

3569-3876 poly A

3877-6063 total backbone

5077-5937 (complementary strand) beta lactamase

(The location of the provided elements and the overall size of the vector are for illustrative purposes only and will depend on the size of the antibody chain insert)

Clones with correctly cloned V _H and V _L sequences were identified and plasmids prepared for expression in suspension culture CHO cells. Expressed 2E7c antibodies were purified from cell culture supernatants by Protein A chromatography on an FPLC system and then tested for binding to Aβ using Biacore ™ technology by ELISA and SPR. This result indicated that the correct 2E7 mouse V region was cloned and expressed, resulting in a functional antibody with properties similar to parental murine antibody 2E7.

Light chain humanization

Human receptor sequences with Genpept ID CAA51135 (SEQ ID NO: 24) and Genbank Accesion No X72467, with 77% identity at the amino acid level (including CDR), were selected as receptor framework. Construct L1 is a graft of murine CDRs from the 2E7 VL domain into this receptor framework.

Heavy chain humanization

The human sequence Genbank accession No M99675 (SEQ ID NO: 21), an allele of the VH3-48 gene with 74% identity to the 2E7 mouse variable heavy chain region at the amino acid level (including CDRs 1 and 2), was loaded with the human JH4 minigene It was selected as the receptor framework. Three humanized variable heavy chain variants were designed based on the M99675 sequence and JH4. H1 is a graft of murine CDRs using the Kabat definition with two additional framework return mutations at positions 93 and 94. Both H2 and H3 are derived from H1 but contain one additional framework mutation that is different in each construct (positions 24 and 48, respectively; see Table 4).

Table 4

Construction of Humanized Heavy and Light Chain DNA

Humanized V regions were resynthesized by construction of overlapping oligos and PCR heavy. Human immunoglobulin signal sequences derived from selected human receptor frameworks and restriction sites for cloning into mammalian expression vectors RLD-bshe and RLN-bshe were included. DNA encoding a humanized V region (H1 (SEQ ID NO: 27), H2 (SEQ ID NO: 29), H3 (SEQ ID NO: 31), L1 (SEQ ID NO: 33)) together with a signal sequence and a restriction site Cloned in frame: H1, H2 and H3 were put into RLD-bshe to encode three full-length human IgG1 Fc mutated heavy chains containing mutations L235A and G237A, full length H1 (SEQ ID NO: 35), full length H2 ( SEQ ID NO: 37) and full length H3 (SEQ ID NO: 39); L1 was cloned in frame with RLN-bshe containing DNA encoding human kappa constant region to generate DNA encoding the full length human kappa light chain (SEQ ID NO: 41).

Representative Examples of Expression of Humanized Heavy and Light Chain Antibody Combinations

CHOK1 cells were transiently transfected in small scale in 6-well plates with all combinations of humanized light and heavy chain DNA constructs: L1 + H1, L1 + H2, L1 + H3 (SEQ ID NOs: 35 + 41, 37 +41, 39 + 41). CHOK1 cells passaged in DMEM F12 with 5% ultra-low IgG fetal bovine serum and 2 mM glutamine were grown confluent in 6-well plates. Confluent cells were transfected with 7.5 μg total DNA: 30 μg transfast lipid (Promega) in Optime Glutamax medium (Invitrogen). Transfected cells were incubated at 37 ° C. with 5% CO ₂ . At 72 hours, the supernatants were recovered and assayed for antibody concentrations and then tested for binding to human Aβ by ELISA. Humanized L1 in combination with three humanized heavy chains all expressed complete antibodies bound to human Aβ.

Humanized antibodies were expressed at large scale with transient CHOK1 cell transfection using liposome delivery of DNA (eg, transmega (Promega)) and expression in culture bottles. To optimize expression levels in transient transfection, the heavy to light chain expression vector DNA ratio was used at 1: 6. Materials from transient transfections were purified using FPLC with ProSepA column or ProSepA HiTrap column.

Evaluation of 2E7 Humanized Variables H1L1, H2L1, and H3L1 in β-amyloid Binding ELISA

2E7 H1L1, H2L1 and H3L1 humanized variants were evaluated for binding to the biotinylated human Αβ peptide (1-40) at the C terminus. Chimera 2E7 was used as a reference. Table 5-7 shows the results for purified batches of various batches from large scale transient transfection.

Table 5

Table 6

Table 7

The results showed a very similar Aβ binding profile for each 2E7-derived humanized variant. Comparison of EC50 values for 2E7c showed that little loss of Aβ binding activity occurred through the humanization process.

Comparison of 2E7 Humanized Variants by Competitive ELISA

The 2E7c chimeric and humanized antibodies H1L1, H2L1 and H3L1 were evaluated for their ability to inhibit binding between human Αβ peptide and parental mouse 2E7 MAb in a competitive ELISA.

Two types of competitive ELISAs were established to compare the Αβ binding activity of the three humanized variants with 2E7 chimeric antibodies. 1) fixed β-amyloid ; Biotinylated human Αβ peptide (1-40) is immobilized on the ELISA plate via streptavidin. Mouse 2E7 antibodies are added at constant concentration following serial dilution of 2E7-derived humanized variant antibodies. Bound mouse 2E7 MAb is detected with anti-mouse IgG conjugate. Table 8 shows the results of the two assays.

Table 8

2) β-amyloid in solution ; A constant concentration of β-amyloid was previously incubated with serially diluted humanized 2E7 antibody variants-a mixture comprising complexed amyloid and free amyloid was added to the wells containing fixed mouse 2E7 MAb for a short time. The amount of free β-amyloid still available for binding to immobilized parent 2E7 MAb was detected. Table 9 shows the results of both assays.

Table 9

All humanized antibody variants inhibited the binding of mouse 2E7 MAb to β-amyloid in a very similar profile. The IC ₅₀ values generated for the H2L1 and H3L1 variants were consistently close to those of the 2E7c chimera (if used), which had the highest inhibitory activity in both assays. However, variant H1L1 showed somewhat reduced inhibitory activity in both assays, which showed a slightly lowered affinity for β-amyloid.

SPR Biacore ™ Analysis of 2E7, 2E7c, H1L1, H2L1, H3L1

Kinetic parameters of recombinant mouse 2E7 MAb, chimeric 2E7c and humanized variants H1L1, H2L1 and H3L1 bound to human beta-amyloid peptides (1-40) and (1-42) were analyzed using Biacore ™ analysis on Biacore ™ 3000. Evaluated. Two different assay formats were used.

Method A

(i) Briefly, <20 resonance units of beta-amyloid 1-40 peptide (biotinylated at the C-terminus) were captured on streptavidin biosensor chips (as used in the case of Table 10A). Antibodies were diluted down in HBS-EP buffer and passed over the streptavidin / beta-amyloid surface at concentrations ranging from 0.001 nM-8 nM (for Table 10A). Two separate runs were performed; Each run was performed on a fresh streptavidin / beta-amyloid surface. Runs 1 and 2 were essentially the same, although there were some differences in the parameters used; Run 1 was performed using a chip surface trapped with 16 RU of beta-amyloid, an antibody concentration of 0.001 nM-8 nM, flow rate of 50 μl / min for 4 min association time and 20 min separation time. It was used as. For run 2, less than 10 RU of beta-amyloid was captured and an antibody concentration of 0.003125 nM-8 nM was used. Flow rate and association time were the same as run 1, but separation time was reduced by 15 minutes.

(ii) Beta amyloids (1-40) and (1-42) were amine coupled to the <20 resonance unit level on different surfaces of the CM5 biosensor chip (as used for Table 10B). Antibodies were diluted down in HBS-EP buffer and passed over the biosensor / beta-amyloid surface at concentrations ranging from 1 nM-64 nM (as used for Table 10B).

Method B

In a second example, the antibody is first subjected to anti-mouse IgG polyclonal antibody surface (for recombinant mouse 2E7 MAb) or Protein A surface (for humanized H2L1) of the CM5 biosensor chip at the level of 1000-2500 resonance units. The assay was reversed in that it captured in the phase. Freshly prepared beta-amyloid (1-40) or (1-42) was diluted down in HBS-EP buffer and passed over the captured-antibody surface at concentrations ranging from 4-500 nM (Tables 10C and 10D).

In both methods, regeneration was performed via a pulse of 100 mM H ₃ PO ₄ , followed by a pulse of 50 mM NaOH for the Table 10A data. The surface was stable and appeared to be unaffected by regeneration. All runs were double quoted for buffer blank injection. The analysis was performed using Biacore ™ analysis software BIAevaluation version 4.1.

result

Antibody was ranked by beta-amyloid binding kinetics data using Method A (i). The data obtained are shown in Table 10A. This indicates that the parental 2E7 Mab has a KD of 36.1 pM for streptavidin-captured beta-amyloid. Chimeric mouse-human antibodies showed a slightly reduced KD of 45.8 pM and humanized constructs ranged from 54 (H2L1) to 93.6 pM (H1L1). In conclusion, this demonstrates that the humanization procedure was very successful and there was little loss of affinity. Additional back mutations introduced for H2 and H3 were small, but had a beneficial effect despite the differences between the H2 and H3 constructs within the standard error for these experiments.

Table 10A

Using Method A (ii) two additional amino acid residues on the C-terminus of beta-amyloid (1-42) do not significantly alter the binding properties of 2E7 and H2L1 compared to beta-amyloid (1-40) It was confirmed that not. The data obtained are shown in Table 10B and corroborate this.

Table 10B

Method B was used to negate the antigenic avidity effects potentially seen in the first assay format. Antigen-binding effects caused by both Fab domains of a single antibody molecule simultaneously bound to two adjacent beta-amyloid molecules (or in the multimeric form beta-amyloid) on the biosensor surface will increase the apparent affinity of binding. . Affinity measurements obtained using Method B are shown in Table 10C.

Table 10C

Evidence that the assay provided true 1: 1 binding affinity indicates that Fab fragments of H2L1 obtained by papain digestion were streptavidin-captured beta-amyloid (1- 40) was obtained when bound with an estimated KD of 2.4 nM.

Using Method B, two additional amino acid residues on the C-terminus of beta-amyloid (1-42) were used to clone the same sequence clone for mouse 2E7 MAb, referred to as 2F11 as compared to beta-amyloid (1-40). It was confirmed that the binding properties were not significantly changed. The data obtained are shown in Table 10D.

Table 10D

In a study similar to the epitope mapping study on 2E7 using the surface plasmon resonance assay described above, H2L1 binds to a peptide comprising amino acids 1-12 of the β-amyloid peptide (Aβ1, SEQ ID NO: 15) but is a β-amyloid peptide. It was similar to 2E7 in that it was not bound to a peptide containing amino acids 2-13 (Aβ2-13, SEQ ID NO: 44).

I ¹²⁵  Activity of H2L1 in the β-amyloid Spill Model

To functionally compare humanized H2L1 with parental mouse monoclonal 2E7, both were tested on the same day with the I ¹²⁵ β-amyloid efflux model described above.

Both H2L1 and 2E7 significantly increased the counts per minute (CPM) in the blood compared to the vehicle control. The CPM of blood radioactivity was as follows (vehicle: 1940 ± 166; 2E7: 10065 ± 1386; H2L1: 10913 ± 1535). The statistics used were ANOVA using post-hoc LSD test. n = 7 vehicle, n = 6 2E7, n = 6 H2L1, p <0.001 for each test compound vs. vehicle.

This data provides further evidence that the humanized H2L1 antibody possessed the functional properties shown in the mouse 2E7 molecule.

Investigation of pharmacokinetics of H2L1 and 2E7

The final half-life of the test antibody was examined in mice. Test antibodies were administered to four mice by 1 hour intravenous infusion to achieve a target dose of 400 μg per mouse. Serial blood samples were taken from each mouse up to 5 days post dosing (except from subsequent analysis as it was clear that one mouse from the 2E7 group did not complete the study and one from the H2L1 group could not be dosed iv). Became). Antibody levels were measured using β-amyloid capture ELISA.

Data analysis showed that humanized antibody H2L1 had a final half-life of approximately 82 hours in mice (Table 11), which is comparable to parental mouse monoclonal antibody 2E7 (approximately 75 hours).

Table 11

#Intermediate value and range

Cmax maximum plasma concentration observed

Tmax The time of maximum plasma concentration observed

CLp total plasma clearance; Capacity / AUC _(0-inf)

The t½ final half-life was determined as the ratio of In 2 / Z (where z is the final phase rate constant; minus weight (after log transformation) for concentration-time pairs occurring after the visually assessed initiation of the final log-linear phase. (unweighted) calculated using linear regression analysis)

Distribution volume at Vss stationary state; CLp x MRT _0-inf

Effect of H2L1 on Peripheral Amyloid Rod in Aged Non-human Primates

A study was conducted in aged cynomolgus monkeys (approximately 15 years old) to investigate exposure response correlations for amyloid / H2L1 complex formation and clearance and subsequent effects on CSF and CNS amyloid levels. Weekly waist CSF (taken under ketamine sedation) and blood samples were collected three weeks before the first dose of H2L1. Immediately after sampling at 3 weeks, animals received placebo (n = 10), 0.1 mg / kg (n = 5), 1 mg / kg (n = 5) or 10 mg / kg (n = 10) of H2L1 every 2 weeks. 12 weeks each week. Blood samples for plasma analysis of H2L1 and total Aβ ₄₂ were obtained weekly. CSF samples were collected every two weeks to quantify Aβ _40/42 . After completion of the dosing period, animals were euthanized for the purpose of brain quantitation and microbleeding investigation of beta-amyloid by the biochemical assay described above. In the lowest dose group (0.1 mg / kg), animals were euthanized in a staggered fashion to assess the effect of dosing termination and the potential time course effects of brain levels as saturation of plasma amyloid pools.

The study was licensed by the Institutional Animal Care and Youth Commission (IACUC) of Maxine Piti Elti, or “Maccine” prior to beginning the experimental phase. The IACUC protocol number was # 08-2006. GSK received Maxine's on-site visit and went through an ethical reconsideration process to determine that this could be tolerated.

Plasma samples were serially diluted from 1:10 to 1: 50000 and added to Aβ ₄₀ coated ELISA plates. Standard curves were drawn in the range of 0-10 μg / ml H 2 L 1 in diluent. After incubation at 4 ° C. overnight, H2L1 was visualized using anti-human IgG horseradish peroxidase (Amersham-diluted 1: 2000 in diluent) and tetramethylbenzidene detection system. After single and repeated iv bolus administration, plasma levels of H2L1 have been shown to increase in a dose dependent fashion. Severe non-linearity was not seen in pharmacokinetics, indicating that for most dosing intervals, an excess molar concentration of H2L1 in plasma was achieved relative to free amyloid levels.

Total Aβ ₄₂ was measured in neat plasma using a commercially available Aβ 1-42 ELISA kit (Innogenetics) according to the manufacturer's instructions, and standard curves were generated in the range of 500-7 pg / ml in kit dilutions. Samples and references were incubated overnight at 4 ° C. and then assayed in duplicate according to kit instructions. It should be noted that, due to the interference of the detection antibodies supplied to the Aβ ₄₂ assay, the kit cannot be used to measure free Aβ ₄₂ levels but measures the apparent 'total' Aβ ₄₂ . There was a dose and concentration dependent increase in Aβ ₄₂ (with stable levels of approximately 300, 125 and 25 pg / ml detected after 10, 1 or 0.1 mg / kg H2L1, respectively). From the analysis, the increase in “total Aβ ₄₂ ” seems to be the result of a significant outflow of amyloid from outside the plasma pool, which appears to be dependent on a H2L1 concentration of> 1 μg / mL, and was not considered to be due to insufficient clearance of the complex. This was evident by the removal rate of total Aβ ₄₂ as well as the change in total level during the dosing interval.

To date, plasma analysis has been achieved and fully analyzed. However, preliminary analysis shows a decreasing trend in CSF and an increase in hippocampus of 'total' Aβ42 (measured as generally disclosed above) after treatment with 10 mg / kg of H2L1.

In some brain sections, the small area of microbleeding was detected as shown by the Percussion Staining method of Perl. This method visualizes ferric iron by producing soluble blue compounds (iron is an essential component of the oxygen-carrying hemoglobin found in red blood cells). However, there was no difference in incidence between vehicle and drug treated animals.

Analysis of Aged Cynomolgus Macaque Monkeys on Beta-amyloid Plaques in the Brain and Total Beta-amyloid in Plasma

Cerebrospinal fluid (CSF) and tissue parameters of human AD were displayed in cynomolgus monkeys. Aged cynomolgus monkeys have been shown to have evidence of amyloid deposition (Covance, The cynomolgus monkey as a model for Alzheimer's disease.In: Buse E, Habermann G, Friderichs-Gromoll S, Kaspereit J, Nowak P and Niggemann K, editors.Poster Presentation at the 44th Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, 6 to 10 March 2005). The possibility of H2L1 inducing an inadequate response (eg meningitis) in the aged brain was investigated in approximately 20 year old ex-breeding female monkeys. In addition, the safety of the test substance, treatment-related microbleeding, neutralization / cleaning rate, irritability, and immune complex disease were also investigated after intravenous administration for 8 weeks at 2 week intervals. In addition, CNS and blood samples were analyzed for levels of Aβ _40/42 .

Study design

Groups of 5 (group 1), 9 (group 2) or 10 (group 3) geriatric female cynomolgus monkeys were treated in 0 (vehicle), vehicle (4 ml / kg) for 8 weeks every 2 weeks. H2L1 was given intravenously by slow bolus administration at 50 or 100 mg / kg / dose. The vehicle consisted of 6.81 mg / mL sodium acetate trihydrate, 0.0186 mg / mL disodium edetate dehydrate, 0.2 mg / mL polysorbate 80, and 10 mg / mL L-arginine base, with a pH of 5.5. Dose levels were selected to investigate dose levels, the intended clinical dose levels, 5 and 10 times.

The following assessments were performed pre-dose, daily (clinical signs, weight, food consumption), 4 weeks, and week before necropsy: live animal observations, body weight, body temperature, hematology, clinical chemistry (including cerebrospinal fluid [CSF] analysis) ), Urine test, and cytokine measurement in CSF. After necropsy, organ weights, macroscopic observations, and microscopic observations of the brain, cervical spinal cord and gross lesions were performed on all animals. Toxic kinetic assessments were performed after each dose.

result

There were no unplanned deaths and no indication of the effect of the test item on the general condition of the animals at the doses administered. Only significant observations in clinical pathology (hematology and serum chemistry) were assumed to be age-related and unrelated to the test substance.

Systemic exposure to H2L1 (measured by AUC _0-τ and C _max ) was largely proportional to dose. For both dose groups, there was no significant change in systemic exposure between the first and fourth dose sampling periods (≥2 fold).

There were no signs of inflammatory responses in the brain detected by CSF-analysis and no gross or microscopic findings suggesting test article influence at necropsy, especially microbleeding or meningitis.

This study is based on German Chemical Law, annex 1 and 2 to §19a Chemikalien Gesetz, June 2002, the OECD Principles of Good Laboratory Practice (revised 1997, issued January 1998) ENV / MC / CHEM (98). ), Conducted in accordance with the Consensus Document "The Application of the OECD Principles of GLP to the Organization and Management of Multi-Site Studies" ENV / JM / MONO (2002) 9]. It is contemplated that studies conducted in compliance with the above regulations and criteria will be accepted by the US FDA.

Analysis of Plaque Rod in the CNS

Left brain hemispheres of vehicle treated cynomolgus macaque monkeys from the study were analyzed by immunohistochemistry. At the level of the middle sulcus containing part of the dentate gyrus and the hippocampus, the parietal section was processed with the wax described above. For immunohistochemistry, sections are labeled with pan-Aβ antibody (1E8, monoclonal antibody raised to Aβ 13-27), or AB42 antibody (20G10, monoclonal antibody that recognizes Aβ x-42). And labeling was as developed above. Visible counts of plaque numbers were performed for each section. Tissues from all five vehicle-treated cynomolgus monkeys showed evidence of parenchymal Aβ plaques. There was no evidence of cerebrovascular labeled Aβ and Aβ in neurons.

Analysis of Beta Amyloid / antibody Complexes in Plasma

On animals at 50 mg / kg (n = 9) or 100 mg / kg (n = 10) H2L1 or plasma samples from vehicle-administered controls (n = 5) at two time points (4 weeks after start of administration and At the end of 8 weeks) biochemical analysis was performed. 100 μl of duplicate samples were analyzed using a commercially available lnnogenetics Aβ 1-42 ELISA kit incubated overnight at 4 ° C. Control samples were analyzed both as pure and as a 1:10 diluent (using supplied diluent), while samples from administered animals were tested as pure and as 1:25. Subsequent absorbance values were analyzed and unknown absorbance values were back-calculated to pg / ml values using standard curves and then corrected for any assay dilution. The total plasma levels of Aβ 42 derived from these samples are shown in Table 12 below (values of pg / ml ± SE); All samples from animals treated with H2L1 contained significantly higher levels of Aβ 42 as compared to controls (p <0.001 by Student's t-test).

Table 12

Reported data were obtained from diluted samples. Since many data scores from pure samples exceeded the highest criterion, or because of sample volume limitations tested only at a single time point, no results were used.

Generation method

Morocloc on a large scale by transfecting or transducing a suitable parental CHO cell line (e.g., CHODG44 or CHOK 1) using an expression vector encoding H2L1 and operably linked to an amplifiable selection marker such as DHFR or glutamine synthetase. Engineered cell lines suitable for generating raw antibodies were generated (see Bebbington and Hentschel DNA Cloning Volume III; A practical approach (edited by Glover DM) (Oxford IRL press, 1987)). To increase expression levels, coding sequences can be codon optimized to avoid cis-acting sequence motifs and extreme GC content (high or low). SEQ ID NOs: 42 and 43 amplify this coding sequence for H2 heavy chain and L1 light chain. Large scale production can be performed and purified in a stirred tank bioreactor using a medium free of animal-derived-components. This may include purification of the recovered product followed by protein-A affinity chromatography and further purification using ion (eg cation) exchange and mixed mode (eg ceramic hydroxyapatite) chromatography unit operations. Final virus / diafiltration steps may be performed following virus removal nanofiltration to ensure that the formulation is suitable for the intended route of administration.

Examples of Pharmaceutical Formulations

ingredient Quantity (per mL)

H2L1 50mg

Sodium Acetate Trihydrate 6.81mg

Polysorbate 80 0.20mg

Arginine Base 10.00mg

Sodium Chloride 3.00mg

Disodium Edetate Dihydrate 0.0186mg

Amount to pH hydrochloric acid 5.5

Make 1.0 mL of water for injection

Fill the nitrogen space part

Amino acid sequence of the V-region and humanized variants of the receptor framework

M99675 heavy chain receptor framework V region amino acid sequence (SEQ ID NO: 21)

M99675 heavy chain receptor framework V region DNA (SEQ ID NO: 22)

CAA51135 Light Chain Receptor Framework V Region Amino Acid Sequence (SEQ ID NO: 24)

CAA51135 Light Chain Receptor Framework V Region DNA (SEQ ID NO: 25)

Humanized heavy chain V region variant H1 amino acid sequence (SEQ ID NO: 26)

Humanized heavy chain V region variant H1 DNA coding sequence (SEQ ID NO: 27)

Humanized heavy chain V region variant H2 amino acid sequence (SEQ ID NO: 28)

Humanized heavy chain V region variant H2 DNA (SEQ ID NO: 29)

Humanized heavy chain V region variant H3 amino acid sequence (SEQ ID NO: 30)

Humanized heavy chain V region variant H3 DNA (SEQ ID NO: 31)

Humanized light chain V region variant L1 amino acid sequence (SEQ ID NO: 32)

Humanized light chain V region variant L1 DNA (SEQ ID NO: 33)

Mature H1 heavy chain amino acid sequence (bolded in Fc mutated double mutant) (SEQ ID NO: 34)

H1 full length DNA (SEQ ID NO: 35)

Mature H2 heavy chain amino acid sequence (in bold Fc mutated double mutant) (SEQ ID NO: 36)

H2 full length DNA (SEQ ID NO: 37)

Mature H3 heavy chain amino acid sequence (Bold mutated double mutated Fc) (SEQ ID NO: 38)

H3 full length DNA (SEQ ID NO: 39)

Mature light chain amino acid sequence (SEQ ID NO: 40)

L 1 full length DNA (SEQ ID NO: 41)

Optimized H2 Heavy Chain DNA (SEQ ID NO: 42)

Optimized L1 light chain DNA (SEQ ID NO: 43)

                                SEQUENCE LISTING <110> Glaxo Group Limited <120> Antibodies    <130> PB61927 <160> 44 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 5 <212> PRT <213> Mouse <400> 1 Asp Asn Gly Met Ala  1 5 <210> 2 <211> 17 <212> PRT <213> Mouse <400> 2 Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val Thr  1 5 10 15 Gly      <210> 3 <211> 6 <212> PRT <213> Mouse <400> 3 Gly Thr Trp Phe Ala Tyr  1 5 <210> 4 <211> 16 <212> PRT <213> Mouse <400> 4 Arg Val Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Thr Tyr Leu His  1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Mouse <400> 5 Lys Val Ser Asn Arg Phe Ser  1 5 <210> 6 <211> 9 <212> PRT <213> Mouse <400> 6 Ser Gln Thr Arg His Val Pro Tyr Thr  1 5 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 7 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Gly Ser Gly Gly Ser Lys  1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 8 Asp Ala Glu Phe Arg His Asp Ser Gly Gly Ser Gly Ser Gly Ser Lys  1 5 10 15 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 9 Asp Ala Glu Phe Arg His Asp Ser Gly Ser Gly Gly Ser Gly Gly Lys  1 5 10 15 <210> 10 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 10 Asp Ala Glu Phe Arg His Asp Gly Ser Gly Gly Ser Gly Gly Ser Lys  1 5 10 15 <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 11 Asp Ala Glu Phe Arg His Gly Ser Gly Gly Ser Gly Gly Ser Gly Lys  1 5 10 15 <210> 12 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 12 Asp Ala Glu Phe Arg Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Lys  1 5 10 15 <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 13 Asp Ala Glu Phe Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Lys  1 5 10 15 <210> 14 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 14 Asp Ala Glu Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Lys  1 5 10 15 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 15 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val Gly Ser Gly Lys  1 5 10 15 <210> 16 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> ACETYLATION <222> (1) ... (0) <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 16 Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Gly Ser Gly Lys  1 5 10 15 <210> 17 <211> 115 <212> PRT <213> Mouse <400> 17 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Arg Lys Gly Pro Glu Trp Ile         35 40 45 Ala Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ala         115 <210> 18 <211> 345 <212> DNA <213> Mouse <400> 18 gaggtgaagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc cctgaaactc 60 tcctgtgcag tctctggatt cactttcagt gacaacggaa tggcgtgggt tcgacaggct 120 ccaaggaagg ggcctgagtg gatagcgttc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tctagagata atgccaagaa taccctgtac 240 ctggaaatga gcagtctgag gtctgaggac acggccatgt actattgtgt aagcgggacc 300 tggtttgctt actggggcca agggactctg gtcactgtct ctgca 345 <210> 19 <211> 112 <212> PRT <213> Mouse <400> 19 Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly  1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Val Ser Gln Ser Leu Leu His Ser             20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Thr                 85 90 95 Arg His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 110 <210> 20 <211> 336 <212> DNA <213> Mouse <400> 20 gatgttgtgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gagttagtca gagcctttta cacagtaatg gatacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaactag acatgttccg 300 tacacgttcg gaggggggac caagctggaa ataaaa 336 <210> 21 <211> 98 <212> PRT <213> Human <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Tyr Ile Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg          <210> 22 <211> 296 <212> DNA <213> Human <400> 22 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtagtagtac catatactac 180 gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaga 296 <210> 23 <211> 15 <212> PRT <213> Human <400> 23 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser  1 5 10 15 <210> 24 <211> 112 <212> PRT <213> Human <400> 24 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly  1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser             20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala                 85 90 95 Leu Gln Thr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 25 <211> 336 <212> DNA <213> Human <400> 25 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300 tggacgttcg gccaagggac caaggtggaa atcaaa 336 <210> 26 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Humanised <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 27 <211> 345 <212> DNA <213> Human <400> 27 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtttcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctca 345 <210> 28 <211> 115 <212> PRT <213> Human <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 29 <211> 345 <212> DNA <213> Human <400> 29 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag tctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtttcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctca 345 <210> 30 <211> 115 <212> PRT <213> Human <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 31 <211> 345 <212> DNA <213> Human <400> 31 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg gatctcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctca 345 <210> 32 <211> 112 <212> PRT <213> Human <400> 32 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly  1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Val Ser Gln Ser Leu Leu His Ser             20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Thr                 85 90 95 Arg His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 110 <210> 33 <211> 336 <212> DNA <213> Human <400> 33 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca gagttagtca gagcctttta cacagtaatg gatacaccta tttacattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaactag acatgttccg 300 tacacgttcg gcggagggac caaggtggaa atcaaa 336 <210> 34 <211> 445 <212> PRT <213> Human <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro         115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val     130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly                 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly             180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys         195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys     210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly Ala Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu                 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys             260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys         275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu     290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser             340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys         355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln     370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln                 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 35 <211> 1335 <212> DNA <213> Human <400> 35 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtttcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctcagcctc caccaagggc 360 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540 agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660 actcacacat gcccaccgtg cccagcacct gaactcgcgg gggcaccgtc agtcttcctc 720 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1080 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320 ctgtctccgg gtaaa 1335 <210> 36 <211> 445 <212> PRT <213> Human <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro         115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val     130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly                 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly             180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys         195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys     210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly Ala Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu                 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys             260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys         275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu     290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser             340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys         355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln     370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln                 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 37 <211> 1335 <212> DNA <213> Human <400> 37 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag tctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtttcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctcagcctc caccaagggc 360 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540 agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660 actcacacat gcccaccgtg cccagcacct gaactcgcgg gggcaccgtc agtcttcctc 720 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1080 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320 ctgtctccgg gtaaa 1335 <210> 38 <211> 445 <212> PRT <213> Human <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly  1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Asn             20 25 30 Gly Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile         35 40 45 Ser Phe Ile Ser Asn Leu Ala Tyr Ser Ile Asp Tyr Ala Asp Thr Val     50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Val Ser Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro         115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val     130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly                 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly             180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys         195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys     210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly Ala Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu                 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys             260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys         275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Val Ser Leu     290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys                 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser             340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys         355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln     370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln                 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn             420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 39 <211> 1335 <212> DNA <213> Human <400> 39 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gacaacggaa tggcgtgggt ccgccaggct 120 ccagggaagg ggctggagtg gatctcattc attagtaatt tggcatatag tatcgactac 180 gcagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgt cagcgggacc 300 tggtttgctt actggggcca gggcacacta gtcacagtct cctcagcctc caccaagggc 360 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 420 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 480 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 540 agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 660 actcacacat gcccaccgtg cccagcacct gaactcgcgg gggcaccgtc agtcttcctc 720 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 780 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 840 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 960 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1020 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 1080 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1260 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320 ctgtctccgg gtaaa 1335 <210> 40 <211> 219 <212> PRT <213> Human <400> 40 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly  1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Val Ser Gln Ser Leu Leu His Ser             20 25 30 Asn Gly Tyr Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser         35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro     50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Thr                 85 90 95 Arg His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu         115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe     130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser                 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu             180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser         195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 <210> 41 <211> 657 <212> DNA <213> Human <400> 41 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca gagttagtca gagcctttta cacagtaatg gatacaccta tttacattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct ataaagtttc caaccgattt 180 tctggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgct ctcaaactag acatgttccg 300 tacacgttcg gcggagggac caaggtggaa atcaaacgta cggtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggacaa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt 657 <210> 42 <211> 1335 <212> DNA <213> Human <400> 42 gaggtgcagc tggtggagtc tggcggcgga ctggtgcagc ctggcggcag cctgagactg 60 agctgtgccg tgtccggctt caccttcagc gacaacggca tggcctgggt gaggcaggcc 120 cctggcaagg gcctggagtg ggtgtccttc atcagcaacc tggcctacag catcgactac 180 gccgacaccg tgaccggcag attcaccatc agccgggaca acgccaagaa cagcctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgt gagcggcacc 300 tggttcgcct actggggcca gggcaccctg gtgaccgtgt ccagcgccag caccaagggc 360 cccagcgtgt tccccctggc ccccagcagc aagagcacca gcggcggcac agccgccctg 420 ggctgcctgg tgaaggacta cttccccgaa ccggtgaccg tgtcctggaa cagcggagcc 480 ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct gtacagcctg 540 agcagcgtgg tgaccgtgcc cagcagcagc ctgggcaccc agacctacat ctgtaacgtg 600 aaccacaagc ccagcaacac caaggtggac aagaaggtgg agcccaagag ctgtgacaag 660 acccacacct gccccccctg ccctgccccc gagctggccg gagcccccag cgtgttcctg 720 ttccccccca agcctaagga caccctgatg atcagcagaa cccccgaggt gacctgtgtg 780 gtggtggatg tgagccacga ggaccctgag gtgaagttca actggtacgt ggacggcgtg 840 gaggtgcaca atgccaagac caagcccagg gaggagcagt acaacagcac ctaccgggtg 900 gtgtccgtgc tgaccgtgct gcaccaggat tggctgaacg gcaaggagta caagtgtaag 960 gtgtccaaca aggccctgcc tgcccctatc gagaaaacca tcagcaaggc caagggccag 1020 cccagagagc cccaggtgta caccctgccc cctagcagag atgagctgac caagaaccag 1080 gtgtccctga cctgcctggt gaagggcttc taccccagcg acatcgccgt ggagtgggag 1140 agcaacggcc agcccgagaa caactacaag accacccccc ctgtgctgga cagcgatggc 1200 agcttcttcc tgtacagcaa gctgaccgtg gacaagagca gatggcagca gggcaacgtg 1260 ttcagctgct ccgtgatgca cgaggccctg cacaatcact acacccagaa gagcctgagc 1320 ctgtcccctg gcaag 1335 <210> 43 <211> 657 <212> DNA <213> Human <400> 43 gacatcgtga tgacccagag ccccctgagc ctgcccgtga cccctggcga gcccgccagc 60 atcagctgta gagtgagcca gagcctgctg cacagcaacg gctacaccta cctgcactgg 120 tatctgcaga agcctggcca gagccctcag ctgctgatct acaaggtgtc caaccggttc 180 agcggcgtgc ctgatagatt cagcggcagc ggctccggca ccgacttcac cctgaagatc 240 agcagagtgg aggccgagga tgtgggcgtg tactactgct cccagaccag acacgtgcct 300 tacacctttg gcggcggaac aaaggtggag atcaagcgta cggtggccgc ccccagcgtg 360 ttcatcttcc cccccagcga tgagcagctg aagagcggca ccgccagcgt ggtgtgtctg 420 ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa tgccctgcag 480 agcggcaaca gccaggagag cgtgaccgag caggacagca aggactccac ctacagcctg 540 agcagcaccc tgaccctgag caaggccgac tacgagaagc acaaggtgta cgcctgtgag 600 gtgacccacc agggcctgtc cagccccgtg accaagagct tcaaccgggg cgagtgc 657 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Peptide <221> MOD_RES <222> (16) ... (0) <223> Biotinylated <400> 44 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His Gly Ser Gly Lys  1 5 10 15

Claims (20)

In a human receptor heavy chain variable region derived from the VH3 gene family, the following CDRs are included: CDRH1: DNGMA (SEQ ID NO: 1) CDRH2: FISNLAYSIDYADTVTG (SEQ ID NO: 2) CDRH3: GTWFAY (SEQ ID NO: 3), In a human receptor light chain variable region derived from the amino acid sequence set forth in SEQ ID NO: 24, the following CDRs are included: CDRL1: RVSQSLLHSNGYTYLH (SEQ ID NO: 4) CDRL2: KVSNRFS (SEQ ID NO: 5) CDRL3: SQTRHVPYT (SEQ ID NO: 6), The framework sequence of the human receptor heavy chain variable region is derived from Framework 4 sequence and SEQ ID NO: 21, wherein the Framework 4 sequence is encoded by human JH4 minigene YFDYWGQGTLVTVSS (SEQ ID NO: 23), A therapeutic antibody that binds to β-amyloid peptide. delete The therapeutic antibody of claim 1, wherein the framework sequence comprises a substitution selected from (i), (ii) or (iii): (i) location Residue 93 V or conservative substituents thereof 94 S or conservative substituents thereof or (ii) location Residue 24 V or conservative substituents thereof 93 V or conservative substituents thereof 94 S or conservative substituents thereof or (iii) location Residue 48 I or a conservative substituent thereof 93 V or conservative substituents thereof 94 S or conservative substituents thereof A therapeutic antibody comprising a V _H chain having the sequence set forth in SEQ ID NO: 26 and a V _L domain having the sequence set forth in SEQ ID NO: 32. A therapeutic antibody comprising a V _H chain having the sequence set forth in SEQ ID NO: 28 and a V _L domain having the sequence set forth in SEQ ID NO: 32. A therapeutic antibody comprising a V _H chain having the sequence set forth in SEQ ID NO: 30 and a V _L domain having the sequence set forth in SEQ ID NO: 32. The therapeutic antibody according to any one of claims 1, 3, 4, 5 or 6, which is an IgG1 isotype. 7. The method of any one of claims 1, 3, 4, 5 or 6, comprising: a) the function of activating complement by traditional pathways; And b) therapeutic antibodies, which essentially lack the function of mediating cytotoxicity of antibody-dependent cells. 8. The therapeutic antibody of claim 7, wherein residues 235 and 237 of the heavy chain constant region are mutated to alanine. The therapeutic antibody of claim 1, wherein the antibody comprises a heavy chain having the sequence set forth in SEQ ID NO: 34, 36 or 38 and a light chain having the sequence set forth in SEQ ID NO: 40. A pharmaceutical composition for treating Alzheimer's disease comprising the therapeutic antibody according to any one of claims 1, 3, 4, 5 or 6. An antibody comprising a V _H domain having the sequence of SEQ ID NO. 17 and a V _L domain having the sequence of SEQ ID NO. delete delete delete delete delete delete delete delete